Deuterated o-sulfated beta lactam hydroxamic acids and deuterated n- sulfated beta lactams

ABSTRACT

Provided herein are deuterated O-sulfated beta-lactam hydroxamic acids and deuterated N-sulfated beta-lactams, pharmaceutical compositions thereof and methods of treating infectious disease with deuterated compounds or pharmaceutical compositions thereof.

This application claims priority under 35 U.S.C. §119 (e) from U.S.Provisional Application Ser. No. 62/322,088, filed Apr. 13, 2016 whichis hereby incorporated by reference in its entirety.

FIELD

Provided herein are deuterated O-sulfated beta-lactam hydroxamic acidsand deuterated N-sulfated beta-lactams, pharmaceutical compositionsthereof and methods of treating infectious disease with deuteratedcompounds or pharmaceutical compositions thereof.

BACKGROUND

Overuse, incorrect use and agricultural use of antibiotics has led tothe emergence of resistant bacteria that are refractory to eradicationby conventional anti-infective agents, such as those based oncarbapenem, cephalosporin or fluoroquinolone architectures. Alarmingly,many of these resistant bacteria are responsible for common infectionsincluding, for example, pneumonia, sepsis, etc.

The dearth of new antibiotic agents, which, inter alia, is due totermination of research and development efforts to develop newantibiotics agents, has exacerbated the above situation. Even at thisdate, when a clear need for novel antibiotic agents has beenestablished, reduced economic incentives and heightened regulatoryrequirements has prevented substantial investment by pharmaceuticalorganizations in this increasingly critical issue in health care.

Failure to provide new agents to treat resistant bacteria threatens themany benefits achieved with antibiotics in the recent past. Accordingly,what is need are novel antibiotic compounds which are effective againstresistant bacteria and are simple to manufacture and use.

SUMMARY

The present application satisfies these and other needs by providingdeuterated O-sulfated beta-lactam hydroxamic acids and deuteratedN-sulfated beta-lactams, which may be used to treat infectious diseases.In one aspect, tigemonam or aztreonam, where any or up to all of thenon-exchangeable hydrogen atoms are substituted with deuterium areprovided.

In another aspect, a compound of structural Formula (I) is provided:

and pharmaceutically acceptable salts, hydrates and solvates thereof,where R₁ and R₂ are independently hydrogen, deuterium, —CH₃, —CD₃, —CD₂Hor —CDH₂, R₃ is hydrogen or deuterium, R₄ is —SO₃H or —OSO₃H, R₅ isheteroaryl or substituted heteroaryl optionally substituted with one ormore deuterium atoms; and R₆ is alkyl, substituted alkyl, aryl,substituted aryl, arylalkyl, substituted arylalkyl, heteroalkyl,substituted heteroalkyl, heteroaryl, substituted heteroaryl,heteroarylalkyl, substituted heteroarylalkyl or —CO₂H, optionallysubstituted with one or more deuterium atoms, provided that at least onenon-exchangeable hydrogen atom is substituted with deuterium.

Also provided are derivatives, including esters, enol ethers, enolesters, metabolites and prodrugs of the compounds described herein.Further provided are pharmaceutical compositions which include thecompounds provided herein and a pharmaceutically acceptable vehicle.

Methods of treating, preventing, or ameliorating symptoms of infectiousdisease in a subject are also presented herein. The methods generallyinvolve administering a therapeutically effective amount of deuteratedO-sulfated beta-lactam hydroxamic acids and/or deuterated N-sulfatedbeta-lactams, or pharmaceutical compositions thereof to the subject.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph which illustrates IV dosing of tigemonam (solid line)and(S,Z)-2-(((1-(2-aminothiazol-4-yl)-2-((2,2-dimethyl-d₆-4-oxo-1-(sulfooxy)azetidin-3-yl)amino)-2-oxoethylidene)amino)oxy)aceticacid (dashed line) in rats where the y axis is plasma concentration(ng/mL) and the x axis is time (hours).

FIG. 2 is a graph which illustrates oral dosing of tigemonam (solidline) and(S,Z)-2-(((1-(2-aminothiazol-4-yl)-2-((2,2-dimethyl-d₆-4-oxo-1-(sulfooxy)azetidin-3-yl)amino)-2-oxoethylidene)amino)oxy)aceticacid (dashed line) in rats where the y axis is plasma concentration(ng/mL) and the x axis is time (hours).

FIG. 3 is a scheme which describes the synthesis of(S,Z)-2-(((1-(2-aminothiazol-4-yl)-2-((2,2-dimethyl-d₆-4-oxo-1-(sulfooxy)azetidin-3-yl)amino)-2-oxoethylidene)amino)oxy)aceticacid.

FIG. 4 is a scheme which describes the synthesis of2-(((Z)-(1-(2-aminothiazol-4-yl)-2-(((2R,3S)-2-d₃-2-methyl-4-oxo-1-(sulfooxy)azetidin-3-yl)amino)-2-oxoethylidene)amino)oxy)aceticacid.

FIG. 5 is a scheme which describes the synthesis of2-(((Z)-(1-(2-aminothiazol-4-yl)-2-(((2S,3S)-2-(d₃-methyl-2-methyl-4-oxo-1-(sulfooxy)azetidin-3-yl)amino)-2-oxoethylidene)amino)oxy)aceticacid.

DETAILED DESCRIPTION Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of ordinary skillin the art to which this invention belongs. In the event that there is aplurality of definitions for a term herein, those in this sectionprevail unless stated otherwise.

“Alkyl” by itself or as part of another substituent, refers to asaturated or unsaturated, branched, straight-chain or cyclic monovalenthydrocarbon radical derived by the removal of one hydrogen atom from asingle carbon atom of a parent alkane, alkene or alkyne. Typical alkylgroups include, but are not limited to, methyl; ethyls such as ethanyl,ethenyl, ethynyl; propyls such as propan-1-yl, propan-2-yl,cyclopropan-1-yl, prop-1-en-1-yl, prop-1-en-2-yl, prop-2-en-1-yl(allyl), cycloprop-1-en-1-yl; cycloprop-2-en-1-yl, prop-1-yn-1-yl,prop-2-yn-1-yl, etc.; butyls such as butan-1-yl, butan-2-yl,2-methyl-propan-1-yl, 2-methyl-propan-2-yl, cyclobutan-1-yl,but-1-en-1-yl, but-1-en-2-yl, 2-methyl-prop-1-en-1-yl, but-2-en-1-yl,but-2-en-2-yl, buta-1,3-dien-1-yl, buta-1,3-dien-2-yl,cyclobut-1-en-1-yl, cyclobut-1-en-3-yl, cyclobuta-1,3-dien-1-yl,but-1-yn-1-yl, but-1-yn-3-yl, but-3-yn-1-yl, etc.; and the like. Theterm “alkyl” is specifically intended to include groups having anydegree or level of saturation, i.e., groups having exclusively singlecarbon-carbon bonds, groups having one or more double carbon-carbonbonds, groups having one or more triple carbon-carbon bonds and groupshaving mixtures of single, double and triple carbon-carbon bonds. Wherea specific level of saturation is intended, the expressions “alkanyl,”“alkenyl,” and “alkynyl” are used. In some embodiments, an alkyl groupcomprises from 1 to 20 carbon atoms (C₁-C₂₀ alkyl). In otherembodiments, an alkyl group comprises from 1 to 10 carbon atoms (C₁-C₁₀alkyl). In still other embodiments, an alkyl group comprises from 1 to 6carbon atoms (C₁-C₆ alkyl).

“Alkanyl” by itself or as part of another substituent, refers to asaturated branched, straight-chain or cyclic alkyl radical derived bythe removal of one hydrogen atom from a single carbon atom of a parentalkane. Typical alkanyl groups include, but are not limited to,methanyl; ethanyl; propanyls such as propan-1-yl, propan-2-yl(isopropyl), cyclopropan-1-yl, etc.; butanyls such as butan-1-yl,butan-2-yl (sec-butyl), 2-methyl-propan-1-yl (isobutyl),2-methyl-propan-2-yl (t-butyl), cyclobutan-1-yl, etc.; and the like.

“Alkenyl” by itself or as part of another substituent, refers to anunsaturated branched, straight-chain or cyclic alkyl radical having atleast one carbon-carbon double bond derived by the removal of onehydrogen atom from a single carbon atom of a parent alkene. The groupmay be in either the cis or trans conformation about the double bond(s).Typical alkenyl groups include, but are not limited to, ethenyl;propenyls such as prop-1-en-1-yl, prop-1-en-2-yl, prop-2-en-1-yl(allyl), prop-2-en-2-yl, cycloprop-1-en-1-yl; cycloprop-2-en-1-yl;butenyls such as but-1-en-1-yl, but-1-en-2-yl, 2-methyl-prop-1-en-1-yl,but-2-en-1-yl, but-2-en-1-yl, but-2-en-2-yl, buta-1,3-dien-1-yl,buta-1,3-dien-2-yl, cyclobut-1-en-1-yl, cyclobut-1-en-3-yl,cyclobuta-1,3-dien-1-yl, etc.; and the like.

“Alkynyl” by itself or as part of another substituent refers to anunsaturated branched, straight-chain or cyclic alkyl radical having atleast one carbon-carbon triple bond derived by the removal of onehydrogen atom from a single carbon atom of a parent alkyne. Typicalalkynyl groups include, but are not limited to, ethynyl; propynyls suchas prop-1-yn-1-yl, prop-2-yn-1-yl, etc.; butynyls such as but-1-yn-1-yl,but-1-yn-3-yl, but-3-yn-1-yl, etc.; and the like.

“Aryl” by itself or as part of another substituent, refers to amonovalent aromatic hydrocarbon group derived by the removal of onehydrogen atom from a single carbon atom of a parent aromatic ringsystem, as defined herein. Typical aryl groups include, but are notlimited to, groups derived from aceanthrylene, acenaphthylene,acephenanthrylene, anthracene, azulene, benzene, chrysene, coronene,fluoranthene, fluorene, hexacene, hexaphene, hexalene, as-indacene,s-indacene, indane, indene, naphthalene, octacene, octaphene, octalene,ovalene, penta-2,4-diene, pentacene, pentalene, pentaphene, perylene,phenalene, phenanthrene, picene, pleiadene, pyrene, pyranthrene,rubicene, triphenylene, trinaphthalene and the like. In someembodiments, an aryl group comprises from 6 to 20 carbon atoms (C₆-C₂₀aryl). In other embodiments, an aryl group comprises from 6 to 15 carbonatoms (C₆-C₁₅ aryl). In still other embodiments, an aryl group comprisesfrom 6 to 15 carbon atoms (C₆-C₁₀ aryl).

“Arylalkyl” by itself or as part of another substituent, refers to anacyclic alkyl group in which one of the hydrogen atoms bonded to acarbon atom, typically a terminal or sp^(a) carbon atom, is replacedwith an aryl group as, as defined herein. Typical arylalkyl groupsinclude, but are not limited to, benzyl, 2-phenylethan-1-yl,2-phenylethen-1-yl, naphthylmethyl, 2-naphthylethan-1-yl,2-naphthylethen-1-yl, naphthobenzyl, 2-naphthophenylethan-1-yl and thelike. Where specific alkyl moieties are intended, the nomenclaturearylalkanyl, arylalkenyl and/or arylalkynyl is used. In someembodiments, an arylalkyl group is (C₆-C₃₀) arylalkyl, e.g., thealkanyl, alkenyl or alkynyl moiety of the arylalkyl group is (C₁-C₁₀)alkyl and the aryl moiety is (C₆-C₂₀) aryl. In other embodiments, anarylalkyl group is (C₆-C₂₀) arylalkyl, e.g., the alkanyl, alkenyl oralkynyl moiety of the arylalkyl group is (C₁-C₈) alkyl and the arylmoiety is (C₆-C₁₂) aryl. In still other embodiments, an arylalkyl groupis (C₆-C₁₅) arylalkyl, e.g., the alkanyl, alkenyl or alkynyl moiety ofthe arylalkyl group is (C₁-C₅) alkyl and the aryl moiety is (C₆-C₁₀)aryl.

“Compounds” refers to compounds encompassed by structural formulaedisclosed herein and includes any specific compounds within theseformulae whose structure is disclosed herein. Compounds may beidentified either by their chemical structure and/or chemical name Whenthe chemical structure and chemical name conflict, the chemicalstructure is determinative of the identity of the compound. Thecompounds described herein may contain one or more chiral centers and/ordouble bonds and therefore, may exist as stereoisomers, such asdouble-bond isomers (i.e., geometric isomers), enantiomers ordiastereomers. Accordingly, the chemical structures depicted hereinencompass all possible enantiomers and stereoisomers of the illustratedcompounds including the stereoisomerically pure form (e.g.,geometrically pure, enantiomerically pure or diastereomerically pure)and enantiomeric and stereoisomeric mixtures. Enantiomeric andstereoisomeric mixtures can be resolved into their component enantiomersor stereoisomers using separation techniques or chiral synthesistechniques well known to the skilled artisan. The compounds may alsoexist in several tautomeric forms including the enol form, the keto formand mixtures thereof. Accordingly, the chemical structures depictedherein encompass all possible tautomeric forms of the illustratedcompounds. The compounds described also include isotopically labeledcompounds where one or more atoms have an atomic mass different from theatomic mass conventionally found in nature. Examples of isotopes thatmay be incorporated into the compounds described herein include, but arenot limited to, ²H, ³H, ¹³C, ¹⁴C, ¹⁵N, ¹⁸O, ¹⁷O, ³⁵S, etc. In general,it should be understood that all isotopes of any of the elementscomprising the compounds described herein may be found in thesecompounds. Compounds may exist in unsolvated or unhydrated forms as wellas solvated forms, including hydrated forms and as N-oxides. In general,compounds may be hydrated, solvated or N-oxides. Certain compounds mayexist in multiple crystalline or amorphous forms. In general, allphysical forms are equivalent for the uses contemplated herein and areintended to be within the scope of the present application. Further, itshould be understood, when partial structures of the compounds areillustrated, that brackets indicate the point of attachment of thepartial structure to the rest of the molecule.

“Deuterium enrichment” refers to the percentage of incorporation ofdeuterium at a given position in a molecule in the place of hydrogen.For example, deuterium enrichment of 1% at a given position means that1% of molecules in a given sample contain deuterium at the specifiedposition. Because the naturally occurring distribution of deuterium isabout 0.0156%, deuterium enrichment at any position in a compoundsynthesized using non-enriched starting materials is about 0.0156%. Thedeuterium enrichment can be determined using conventional analyticalmethods known to one of ordinary skill in the art, including massspectrometry and nuclear magnetic resonance spectroscopy.

“Is/are deuterium,” when used to describe a given position in a moleculesuch as R₁-R₃₀ or the symbol “D”, when used to represent a givenposition in a drawing of a molecular structure, means that the specifiedposition is enriched with deuterium above the naturally occurringdistribution of deuterium. The same is true of the term “containsdeuterium,” which is often used to refer to methyl groups which may bemono-, di- or trideuterated (e.g., such groups may be —CH₂D, —CD₂H, and—CD₃, wherein each position denoted D is enriched with deuterium abovethe naturally occurring distribution of deuterium). In some embodimentsdeuterium enrichment is no less than about 1%, in others no less thanabout 5%, in others no less than about 10%, in another no less thanabout 20%, in another no less than about 50%, in others no less thanabout 70%, in others no less than about 80%, in others no less thanabout 90%, or in others no less than about 98% of deuterium at thespecified position.

“Heteroalkyl,” “Heteroalkanyl,” “Heteroalkenyl” and “Heteroalkynyl” bythemselves or as part of other substituents, refer to alkyl, alkanyl,alkenyl and alkynyl groups, respectively, in which one or more of thecarbon atoms (and optionally any associated hydrogen atoms), are each,independently of one another, replaced with the same or differentheteroatoms or heteroatomic groups. Typical heteroatoms or heteroatomicgroups which can replace the carbon atoms include, but are not limitedto, —O—, —S—, —N—, —Si—, —NH—, —S(O)—, —S(O)₂—, —S(O)NH—, —S(O)₂NH— andthe like and combinations thereof. The heteroatoms or heteroatomicgroups may be placed at any interior position of the alkyl, alkenyl oralkynyl groups. Typical heteroatomic groups which can be included inthese groups include, but are not limited to, —O—, —S—, —O—O—, —S—S—,—O—S—, —NR⁵⁰¹R⁵⁰²—, ═N—N═, —N═N—, —N═N—NR⁵⁰³R⁵⁰⁴, —PR⁵⁰⁵—, —P(O)₂—,—POR⁵⁰⁶—, —O—P(O)₂—, —SO—, —SO₂—, —SnR⁵⁰⁷R⁵⁰⁸— and the like, where R⁵⁰¹,R⁵⁰², R⁵⁰³, R⁵⁰⁴, R⁵⁰⁵, R⁵⁰⁶, R⁵⁰⁷ and R⁵⁰⁸ are independently hydrogen,alkyl, substituted alkyl, aryl, substituted aryl, arylalkyl, substitutedarylalkyl, cycloalkyl, substituted cycloalkyl, cycloheteroalkyl,substituted cycloheteroalkyl, heteroalkyl, substituted heteroalkyl,heteroaryl, substituted heteroaryl, heteroarylalkyl or substitutedheteroarylalkyl.

“Heteroaryl” by itself or as part of another substituent, refers to amonovalent heteroaromatic radical derived by the removal of one hydrogenatom from a single atom of a parent heteroaromatic ring systems, asdefined herein. Typical heteroaryl groups include, but are not limitedto, groups derived from acridine, β-carboline, chromane, chromene,cinnoline, furan, imidazole, indazole, indole, indoline, indolizine,isobenzofuran, isochromene, isoindole, isoindoline, isoquinoline,isothiazole, isoxazole, naphthyridine, oxadiazole, oxazole, perimidine,phenanthridine, phenanthroline, phenazine, phthalazine, pteridine,purine, pyran, pyrazine, pyrazole, pyridazine, pyridine, pyrimidine,pyrrole, pyrrolizine, quinazoline, quinoline, quinolizine, quinoxaline,tetrazole, thiadiazole, thiazole, thiophene, triazole, xanthene, and thelike. In some embodiments, the heteroaryl group comprises from 5 to 20ring atoms (5-20 membered heteroaryl). In other embodiments, theheteroaryl group comprises from 5 to 10 ring atoms (5-10 memberedheteroaryl). Exemplary heteroaryl groups include those derived fromfuran, thiophene, pyrrole, benzothiophene, benzofuran, benzimidazole,indole, pyridine, pyrazole, quinoline, imidazole, oxazole, isoxazole andpyrazine.

“Heteroarylalkyl” by itself or as part of another substituent refers toan acyclic alkyl group in which one of the hydrogen atoms bonded to acarbon atom, typically a terminal or spa carbon atom, is replaced with aheteroaryl group. Where specific alkyl moieties are intended, thenomenclature heteroarylalkanyl, heteroarylakenyl and/orheteroarylalkynyl is used. In some embodiments, the heteroarylalkylgroup is a 6-21 membered heteroarylalkyl, e.g., the alkanyl, alkenyl oralkynyl moiety of the heteroarylalkyl is (C₁-C₆) alkyl and theheteroaryl moiety is a 5-15-membered heteroaryl. In other embodiments,the heteroarylalkyl is a 6-13 membered heteroarylalkyl, e.g., thealkanyl, alkenyl or alkynyl moiety is (C₁-C₃) alkyl and the heteroarylmoiety is a 5-10 membered heteroaryl.

“Hydrates” refers to incorporation of water into to the crystal latticeof a compound described herein, in stoichiometric proportions, resultingin the formation of an adduct. Methods of making hydrates include, butare not limited to, storage in an atmosphere containing water vapor,dosage forms that include water, or routine pharmaceutical processingsteps such as, for example, crystallization (i.e., from water or mixedaqueous solvents), lyophilization, wet granulation, aqueous filmcoating, or spray drying. Hydrates may also be formed, under certaincircumstances, from crystalline solvates upon exposure to water vapor,or upon suspension of the anhydrous material in water. Hydrates may alsocrystallize in more than one form resulting in hydrate polymorphism. Seee.g., (Guillory, K., Chapter 5, pp. 202-205 in Polymorphism inPharmaceutical Solids, (Brittain, H. ed.), Marcel Dekker, Inc., NewYork, N.Y., 1999). The above methods for preparing hydrates are wellwithin the ambit of those of skill in the art, are completelyconventional and do not require any experimentation beyond what istypical in the art. Hydrates may be characterized and/or analyzed bymethods well known to those of skill in the art such as, for example,single crystal X-ray diffraction, X-ray powder diffraction, Polarizingoptical microscopy, thermal microscopy, thermogravimetry, differentialthermal analysis, differential scanning calorimetry, IR spectroscopy,Raman spectroscopy and NMR spectroscopy. (Brittain, H., Chapter 6, pp.205-208 in Polymorphism in Pharmaceutical Solids, (Brittain, H. ed.),Marcel Dekker, Inc. New York, 1999). In addition, many commercialcompanies routine offer services that include preparation and/orcharacterization of hydrates such as, for example, HOLODIAG, PharmaparcII, Voie de l'Innovation, 27 100 Val de Reuil, France(http://www.holodiag.com).

“Parent Aromatic Ring System” refers to an unsaturated cyclic orpolycyclic ring system having a conjugated π electron system.Specifically included within the definition of “parent aromatic ringsystem” are fused ring systems in which one or more of the rings arearomatic and one or more of the rings are saturated or unsaturated, suchas, for example, fluorene, indane, indene, phenalene, etc. Typicalparent aromatic ring systems include, but are not limited to,aceanthrylene, acenaphthylene, acephenanthrylene, anthracene, azulene,benzene, chrysene, coronene, fluoranthene, fluorene, hexacene,hexaphene, hexalene, as-indacene, s-indacene, indane, indene,naphthalene, octacene, octaphene, octalene, ovalene, penta-2,4-diene,pentacene, pentalene, pentaphene, perylene, phenalene, phenanthrene,picene, pleiadene, pyrene, pyranthrene, rubicene, triphenylene,trinaphthalene and the like.

“Preventing” or “prevention” refers to a reduction in risk of acquiringa disease or disorder (i.e., causing at least one of the clinicalsymptoms of the disease not to develop in a patient that may be exposedto or predisposed to the disease but does not yet experience or displaysymptoms of the disease). In some embodiments, “preventing” or“prevention” refers to reducing symptoms of the disease by taking thecompound in a preventative fashion. The application of a therapeutic forpreventing or prevention of a disease of disorder is known as‘prophylaxis.’ In some embodiments, the compounds provided hereinprovide superior prophylaxis because of lower long term side effectsover long time periods.

“Prodrug” refers to a derivative of a drug molecule that requires atransformation within the body to release the active drug. Prodrugs arefrequently (though not necessarily) pharmacologically inactive untilconverted to the parent drug.

“Salt” refers to a salt of a compound, which possesses the desiredpharmacological activity of the parent compound. Such salts include: (1)acid addition salts, formed with inorganic acids such as hydrochloricacid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, andthe like; or formed with organic acids such as acetic acid, propionicacid, hexanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvicacid, lactic acid, malonic acid, succinic acid, malic acid, maleic acid,fumaric acid, tartaric acid, citric acid, benzoic acid,3-(4-hydroxybenzoyl) benzoic acid, cinnamic acid, mandelic acid,methanesulfonic acid, ethanesulfonic acid, 1,2-ethane-disulfonic acid,2-hydroxyethanesulfonic acid, benzenesulfonic acid,4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid,4-toluenesulfonic acid, camphorsulfonic acid,4-methylbicyclo[2.2.2]-oct-2-ene-1-carboxylic acid, glucoheptonic acid,3-phenylpropionic acid, trimethylacetic acid, tertiary butylacetic acid,lauryl sulfuric acid, gluconic acid, glutamic acid, hydroxynaphthoicacid, salicylic acid, stearic acid, muconic acid, and the like; or (2)salts formed when an acidic proton present in the parent compound isreplaced by a metal ion, e.g., an alkali metal ion, an alkaline earthion, or an aluminum ion; or coordinates with an organic base such asethanolamine, diethanolamine, triethanolamine, N-methylglucamine and thelike. In some embodiments, the salt is pharmaceutically acceptable.

“Solvates” refers to incorporation of solvents into to the crystallattice of a compound described herein, in stoichiometric proportions,resulting in the formation of an adduct. Methods of making solvatesinclude, but are not limited to, storage in an atmosphere containing asolvent, dosage forms that include the solvent, or routinepharmaceutical processing steps such as, for example, crystallization(i.e., from solvent or mixed solvents) vapor diffusion, etc. Solvatesmay also be formed, under certain circumstances, from other crystallinesolvates or hydrates upon exposure to the solvent or upon suspensionmaterial in solvent. Solvates may crystallize in more than one formresulting in solvate polymorphism. See e.g., (Guillory, K., Chapter 5,pp. 205-208 in Polymorphism in Pharmaceutical Solids, (Brittain, H.ed.), Marcel Dekker, Inc., New York, N.Y., 1999)). The above methods forpreparing solvates are well within the ambit of those of skill in theart, are completely conventional do not require any experimentationbeyond what is typical in the art. Solvates may be characterized and/oranalyzed by methods well known to those of skill in the art such as, forexample, single crystal X-ray diffraction, X-ray powder diffraction,Polarizing optical microscopy, thermal microscopy, thermogravimetry,differential thermal analysis, differential scanning calorimetry, IRspectroscopy, Raman spectroscopy and NMR spectroscopy. (Brittain, H.,Chapter 6, pp. 205-208 in Polymorphism in Pharmaceutical Solids,(Brittain, H. ed.), Marcel Dekker, Inc. New York, 1999). In addition,many commercial companies routine offer services that includepreparation and/or characterization of solvates such as, for example,HOLODIAG, Pharmaparc II, Voie de l'Innovation, 27 100 Val de Reuil,France (http://www.holodiag.com).

“Substituted” when used to modify a specified group or radical, meansthat one or more hydrogen atoms of the specified group or radical areeach, independently of one another, replaced with the same or differentsubstituent(s). Substituent groups useful for substituting saturatedcarbon atoms in the specified group or radical include, but are notlimited to —R^(a), halo, —O⁻, ═O, —OR^(b), —SR^(b), —S⁻, ═S,—NR^(c)R^(c), ═NR^(b), ═N—OR^(b), trihalomethyl, —CF₃, —CN, —OCN, —SCN,—NO, —NO₂, ═N₂, —N₃, —S(O)₂R^(b), —S(O)₂NR^(b), —S(O)₂O⁻, —S(O)₂OR^(b),—OS(O)₂R^(b), —OS(O)₂O⁻, —OS(O)₂OR^(b), —P(O)(O⁻)₂, —P(O)(OR^(b))(O⁻),—P(O)(OR^(b))(OR^(b)), —C(O)R^(b), —C(S)R^(b), —C(NR^(b))R^(b), —C(O)O⁻,—C(O)OR^(b), —C(S)OR^(b), —C(O)NR^(c)R^(c), —C(NR^(b))NR^(c)R^(c),—OC(O)R^(b), —OC(S)R^(b), —OC(O)O⁻, —OC(O)OR^(b), —OC(S)OR^(b),—NR^(b)C(O)R^(b), —NR^(b)C(S)R^(b), —NR^(b)C(O)O⁻, —NR^(b)C(O)OR^(b),—NR^(b)C(S)OR^(b), —NR^(b)C(O)NR^(c)R^(c), —NR^(b)C(NR^(b))R^(b) and—NR^(b)C(NR^(b))NR^(c)R^(c), where R^(a) is selected from the groupconsisting of alkyl, cycloalkyl, heteroalkyl, cycloheteroalkyl, aryl,arylalkyl, heteroaryl and heteroarylalkyl; each R^(b) is independentlyhydrogen or R^(a); and each W is independently R^(b) or alternatively,the two R^(c)'s are taken together with the nitrogen atom to which theyare bonded form a 4-, 5-, 6- or 7-membered cycloheteroalkyl which mayoptionally include from 1 to 4 of the same or different additionalheteroatoms selected from the group consisting of O, N and S. Asspecific examples, —NR^(c)R^(c) is meant to include —NH₂, —NH-alkyl,N-pyrrolidinyl and N-morpholinyl.

Similarly, substituent groups useful for substituting unsaturated carbonatoms in the specified group or radical include, but are not limited to,—R^(a), halo, —O⁻, —OR^(b), —SR^(b), —S⁻, —NR^(c)R^(c), trihalomethyl,—CF₃, —CN, —OCN, —SCN, —NO, —NO₂, —N₃, —S(O)₂R^(b), —S(O)₂O⁻,—S(O)₂OR^(b), —OS(O)₂R^(b), —OS(O)₂O⁻, —OS(O)₂OR^(b), —P(O)(O⁻)₂,—P(O)(OR^(b))(O⁻), —P(O)(OR^(b))(OR^(b)), —C(O)R^(b), —C(S)R^(b),—C(NR^(b))R^(b), —C(O)O⁻, —C(O)OR^(b), —C(S)OR^(b), —C(O)NR^(c)R^(c),—C(NR^(b))NR^(c)R^(c), —OC(O)R^(b), —OC(S)R^(b), —OC(O)O⁻, —OC(O)OR^(b),—OC(S)OR^(b), —NR^(b)C(O)R^(b), —NR^(b)C(S)R^(b), —NR^(b)C(O)O⁻,—NR^(b)C(O)OR^(b), —NR^(b)C(S)OR^(b), —NR^(b)C(O)NR^(c)R^(c),—NR^(b)C(NR^(b))R^(b) and —NR^(b)C(NR^(b))NR^(c)R^(c), where R^(a),R^(b) and R^(c) are as previously defined.

Substituent groups useful for substituting nitrogen atoms in heteroalkyland cycloheteroalkyl groups include, but are not limited to, —R^(a),—O⁻, —OR^(b), —SR^(b), —S⁻, —NR^(c)R^(c), trihalomethyl, —CF₃, —CN, —NO,—NO₂, —S(O)₂R^(b), —S(O)₂O⁻, —S(O)₂OR^(b), —OS(O)₂R^(b), —OS(O)₂O⁻,—OS(O)₂OR^(b), —P(O)(O⁻)₂, —P(O)(OR^(b))(O⁻), —P(O)(OR^(b))(OR^(b)),—C(O)R^(b), —C(S)R^(b), —C(NR^(b))R^(b), —C(O)OR^(b), —C(S)OR^(b),—C(O)NR^(c)R^(c), —C(NR^(b))NR^(c)R^(c), —OC(O)R^(b), —OC(S)R^(b),—OC(O)OR^(b), —OC(S)OR^(b), —NR^(b)C(O)R^(b), —NR^(b)C(S)R^(b),—NR^(b)C(O)OR^(b), —NR^(b)C(S)OR^(b), —NR^(b)C(O)NR^(c)R^(c),—NR^(b)C(NR^(b))R^(b) and —NR^(b)C(NR^(b))NR^(c)R^(c), where R^(a),R^(b) and R^(c) are as previously defined.

Substituent groups from the above lists useful for substituting otherspecified groups or atoms will be apparent to those of skill in the art.The substituents used to substitute a specified group can be furthersubstituted, typically with one or more of the same or different groupsselected from the various groups specified above. In some embodiments,substituents are limited to the groups above.

“Subject,” “individual” or “patient” is used interchangeably herein andrefers to a vertebrate, preferably a mammal. Mammals include, but arenot limited to, murines, rodents, simians, humans, farm animals, sportanimals and pets.

“Treating” or “treatment” of any disease or disorder refers, in someembodiments, to ameliorating the disease or disorder (i.e., arresting orreducing the development of the disease or at least one of the clinicalsymptoms thereof). Treatment may also be considered to includepreemptive or prophylactic administration to ameliorate, arrest orprevent the development of the disease or at least one of the clinicalsymptoms. Treatment can also refer to the lessening of the severityand/or the duration of one or more symptoms of a disease or disorder. Ina further feature, the treatment rendered has lower potential for longterm side effects over multiple years. In other embodiments “treating”or “treatment” refers to ameliorating at least one physical parameter,which may not be discernible by the patient. In yet other embodiments,“treating” or “treatment” refers to inhibiting the disease or disorder,either physically, (e.g., stabilization of a discernible symptom),physiologically, (e.g., stabilization of a physical parameter) or both.In yet other embodiments, “treating” or “treatment” refers to delayingthe onset of the disease or disorder.

“Therapeutically effective amount” means the amount of a compound that,when administered to a patient for treating a disease, is sufficient toeffect such treatment for the disease. The “therapeutically effectiveamount” will vary depending on the compound, the disease and itsseverity and the age, weight, adsorption, distribution, metabolism andexcretion etc., of the patient to be treated.

“Vehicle” refers to a diluent, excipient or carrier with which acompound is administered to a subject. In some embodiments, the vehicleis pharmaceutically acceptable.

It should be noted that there are alternative ways of implementing thepresent invention. Accordingly, the present embodiments are to beconsidered as illustrative and not restrictive, and the invention is notto be limited to the details given herein, but may be modified withinthe scope and equivalents of the appended claims.

Compounds

Provided herein are deuterated O-sulfated beta-lactam hydroxamic acidsand deuterated N-sulfated beta-lactams, which may be used to treatinfectious diseases. Deuteration of O-sulfated beta-lactam hydroxamicacids and N-sulfated beta-lactams, may, in some instances, increase theanti-infectious activity and/or alter important pharmacologicalproperties, such as, for example, toxicity, clearance, blood levels orbioavailability of the compounds described herein in a beneficialmanner.

In some embodiments, a compound of structural Formula (I) is provided:

or pharmaceutically acceptable salts, hydrates, solvates and polymorphsthereof, where R₁ and R₂ are independently hydrogen, deuterium, —CH₃,—CD₃, —CD₂H or —CDH, R₃ is hydrogen or deuterium, R₄ is —SO₃H or —OSO₃H,R₅ is heteroaryl or substituted heteroaryl optionally substituted withone or more deuterium atoms; and R₆ is alkyl, substituted alkyl, aryl,substituted aryl, arylalkyl, substituted arylalkyl, heteroalkyl,substituted heteroalkyl, heteroaryl, substituted heteroaryl,heteroarylalkyl, substituted heteroarylalkyl or —CO₂H, optionallysubstituted with one or more deuterium atoms, provided that at least onenon-exchangeable hydrogen atom is substituted with deuterium.

In certain embodiments are provided compounds as disclosed herein,wherein at least one of R₁-R₃₀ independently has deuterium enrichment ofno less than about 1% . In certain embodiments are provided compounds asdisclosed herein, wherein at least one of R₁-R₃₀ independently hasdeuterium enrichment of no less than about 10%. In certain embodimentsare provided compounds as disclosed herein, wherein at least one ofR₁-R₃₀ independently has deuterium enrichment of no less than about 50%.In certain embodiments are provided compounds as disclosed herein,wherein at least one of R₁-R₃₀ independently has deuterium enrichment ofno less than about 90%. In certain embodiments are provided compounds asdisclosed herein, wherein at least one of R₁-R₃₀ independently hasdeuterium enrichment of no less than about 95%. In certain embodimentsare provided compounds as disclosed herein, wherein at least one ofR₁-R₃₀ independently has deuterium enrichment of no less than about 98%.

In certain embodiments are provided compounds as disclosed hereinwherein each position represented as D has deuterium enrichment of noless than about 1%. In certain embodiments are provided compounds asdisclosed herein wherein each position represented as D has deuteriumenrichment of no less than about 10%. in certain embodiments areprovided compounds as disclosed herein wherein each position representedas D has deuterium enrichment of no less than about 50%. In certainembodiments are provided compounds as disclosed herein wherein eachposition represented as D has deuterium enrichment of no less than about90%. In certain embodiments are provided compounds as disclosed hereinwherein each position represented as D has deuterium enrichment of noless than about 95%. In certain embodiments are provided compounds asdisclosed herein wherein each position represented as D has deuteriumenrichment of no less than about 98%.

In certain embodiments, the deuterated compounds disclosed hereinmaintain the beneficial aspects of the corresponding non-isotopicallyenriched molecules, while substantially increasing the maximum tolerateddose, increasing bioavailability, increasing blood levels, decreasingtoxicity, increasing the half-life (T_(1/2)), lowering the maximumplasma concentration (C_(max)) of the minimum efficacious dose (MED),lowering the efficacious dose and thus decreasing thenon-mechanism-related toxicity, and/or lowering the probability ofdrug-drug interactions. In particular embodiments, the deuteratedcompounds disclosed herein maintain the beneficial aspects of thecorresponding non-isotopically enriched molecules, while substantiallyincreasing oral bioavailability.

In some embodiments, if R₁ and R₂ are both —CH₃, then either R₃ isdeuterium or R₅ or R₆ is substituted with at least one deuterium. Inother embodiments, if R₁ or R₂ is hydrogen or deuterium then the otherof R₁ or R₂ is not hydrogen or deuterium. In still other embodiments, ifR₁ or R₂ are independently hydrogen or —CH₃, then either R₃ is deuteriumor R₅ or R₆ is substituted with at least one deuterium. In still otherembodiments, R₁ and R₂ are not both —CH₃. In still other embodiments, R₁and R₂ are not independently both hydrogen or —CH₃. In still otherembodiments, R₆ is alkyl, substituted alkyl, aryl, substituted aryl,arylalkyl, substituted arylalkyl, heteroalkyl, substituted heteroalkylor —CO₂H.

In any of the above embodiments, R₅ is

and R₆ is selected from the fragments listed in Table 1, below, wherethe crossed line is the point of attachment of R₆ to the oxime oxygen.

In some embodiments, R₁ and R₂ are CD₃, R₃ is H, R₄ is —OSO₃H, R₅ is

and R₆ is selected from the fragments listed in Table 1, below, wherethe crossed line is the point of attachment of R₆ to the oxime oxygen.

In some embodiments, R₁ is CD₃, R₂ and R₃ are H, R₄ is —SO₃H, R₅ is

and R₆ is selected from the fragments listed in Table 1, below, wherethe crossed line is the point of attachment of R₆ to the oxime oxygen.

TABLE 1

—Me —Et —CO₂H —CH₂C(O)NHOH —CH₂C(O)NHOCPh₃ —CH₂C(O)NHOCHPh₂

—CH₂CO₂CHPh₂ —CH₂CO₂CMe₃ or —CH(Me)₂.

It should be specifically noted that any or all of the non-exchangeablehydrogen atoms of the R₆ substituent, which are depicted above, may besubstituted with deuterium and hence are within the scope of the presentapplication.

In some of the above embodiments of Formula (I), R₅ is

and R₆ is —CH₂CO₂H, —CD₂CO₂H or —CHDCO₂H. In other of the aboveembodiments, R₅ is

and R₆ is —CR₇R₈CO₂H, where R₇ and R₈ are independently —CH₃, —CD₃,—CD₂H or —CDH₂.

In some of the above embodiments, R₁ and R₂ are —CD₃. In other of theabove embodiments, one of R₁ and R₂ is —CH₃ and the other of R₁ and R₂is —CD₃. In still other of the above embodiments, R₁ is hydrogen and R₂is —CH₃, —CD₃, —CD₂H or —CDH₂. In still other of the above embodiments,R₁ is deuterium and R₂ is—CH₃, —CD₃, —CD₂H or —CDH₂. In still other ofthe above embodiments, R₂ is hydrogen and R₁ is —CH₃, —CD₃, —CD₂H or—CDH₂. In still other of the above embodiments, R₂ is deuterium and R₁is —CH₃, —CD₃, —CD₂H or —CDH₂. In still other of the above embodiments,R₃ is hydrogen and R₄ is —SO₃H. In still other of the above embodiments,R₃ is deuterium and R₄ is —SO₃H. In still other of the aboveembodiments, R₃ is hydrogen and R₄ is —OSO₃H. In still other of theabove embodiments, R₃ is deuterium and R₄ is —OSO₃H.

Of particular interest are deuterated derivatives of tigemonam andaztreonam, the parent structures of which are depicted below. It shouldbe noted that any or all of the non-exchangeable hydrogen atoms intigemonam or aztreonam may be substituted with deuterium and hence arewithin the scope of the present invention.

In some embodiments, a compound of structural Formula (II) is provided:

-   or pharmaceutically acceptable salts, hydrates, solvates and    polymorphs thereof, wherein:-   X is —CR₉R₁₀R₁₁;-   Y is —CR₁₂R₁₃R₁₄; and-   R₉-R₁₈ are independently hydrogen or deuterium, provided that at    least one of R₉-R₁₈ is deuterium.

In some embodiments, R₉-R₁₄ are deuterium. In other embodiments, R₁₅-R₁₈are deuterium. In still other embodiments, R₉-R₁₈ are deuterium. Instill other embodiments, R₉-R₁₁ are deuterium. In still otherembodiments, R₁₂-R₁₄ are deuterium. In still other embodiments, R₉-R₁₁and R₁₅-R₁₈ are deuterium. In still other embodiments, R₁₂-R₁₄ andR₁₅-R₁₈ are deuterium. In still other embodiments, R₉-R₁₁ and R₁₇-R₁₈are deuterium. In still other embodiments, R₁₂-R₁₄ and R₁₇-R₁₈ aredeuterium. In still other embodiments, R₉-R₁₄ and R₁₇-R₁₈ are deuterium.

In some embodiments, R₉-R₁₄ are deuterium and R₁₅-R₁₈ are hydrogen. Inother embodiments, R₉-R₁₄ are hydrogen and R₁₅-R₁₈ are deuterium. Instill other embodiments, R₉-R₁₁ are deuterium and R₁₅-R₁₈ are hydrogen.In still other embodiments, R₁₂-R₁₄ are deuterium, R₉-R₁₁ are hydrogenand R₁₅-R₁₈ are hydrogen. In still other embodiments, R₉-R₁₁ and R₁₅-R₁₈are deuterium and R₁₂-R₁₄ are hydrogen. In still other embodiments,R₁₂-R₁₄ and R₁₅-R₁₈ are deuterium and R₉-R₁₁ are hydrogen. In stillother embodiments, R₉-R₁₁ and R₁₇-R₁₈ are deuterium and R₁₂-R₁₆ arehydrogen. In still other embodiments, R₁₂-R₁₄ and R₁₇-R₁₈ are deuteriumand R₉-R₁₁ are hydrogen and R₁₅-R₁₆ are hydrogen. In still otherembodiments, R₉-R₁₄ and R₁₇-R₁₈ are deuterium and R₁₅-R₁₆.

In some embodiments, a compound of structural Formula (III) is provided:

-   -   or pharmaceutically acceptable salts, hydrates, solvates and        polymorphs thereof, wherein:    -   Z is —CR₂₀R₂₁R₂₂;    -   T is —CR₂₅R₂₆R₂₇;    -   T is —CR₂₈R₂₉R₃₀; and    -   R₁₉-R₃₀ are independently hydrogen or deuterium, provided that        at least one of R₁₉-R₃₀ is deuterium.

In some embodiments, R₁₉-R₃₀ are deuterium. In other embodiments,R₂₅-R₃₀ and R₂₀-R₂₂ are deuterium. In still other embodiments, R₂₅-R₃₀and R₁₉ are deuterium. In still other embodiments, R₂₅-R₃₀ aredeuterium. In still other embodiments, R₂₉-R₂₂ deuterium. In still otherembodiments, R₂₅-R_(30,) R₂₀-R₂₂ and R₁₉ are deuterium. In still otherembodiments, R₂₀-R₂₂ are deuterium. In still other embodiments, R₁₉ isdeuterium. In still other embodiments, R₂₃-R₂₄ are deuterium.

In some embodiments, R₂₅-R₃₀ and R₂₀-R₂₂ are deuterium and R₁₉-R₂₁ andR₂₃-R₂₄ are hydrogen. In still other embodiments, R₂₅-R₃₀ and R₁₉ aredeuterium and R₂₀-R₂₄ are hydrogen. In still other embodiments, R₂₅-R₃₀are deuterium and R₁₉-R₂₄ are hydrogen. In still other embodiments,R₁₉-R₂₂ are deuterium and R₂₄-R₃₀ are hydrogen. In still otherembodiments, R₂₅-R_(30,) R₂₀-R₂₂ and R₁₉ are deuterium and R₂₃-R₂₄ arehydrogen. In still other embodiments, R₂₀-R₂₂ are deuterium and R₂₃-R₃₀and R₁₉ are hydrogen are hydrogen. In still other embodiments, R₁₉ isdeuterium and R₂₀-R₃₀ are hydrogen. In still other embodiments, R₂₃-R₂₄are deuterium and R₁₉-R₂₂ are hydrogen and R₂₅-R₃₀ are hydrogen.

Some exemplary compounds of Formulae (I), (II) and (III), which aredeuterated aztreonam and tigemonam derivatives are shown in Table 2,below.

Other deuterated aztreonam and tigemonam structures, which are notdepicted above will be immediately obvious to the skilled artisan andare also within the scope of the present application.

Isotopic hydrogen can be introduced into compounds disclosed herein bysynthetic techniques that employ deuterated reagents, wherebyincorporation rates are predetermined; and/or by exchange techniques,wherein incorporation rates are determined by equilibrium conditions,and may be highly variable depending on the reaction conditions.Synthetic techniques, where deuterium is directly and specificallyinserted by deuterated reagents of known isotopic content, may yieldhigh deuterium abundance, but can be limited by the chemistry required.Exchange techniques, on the other hand, may yield lower deuteriumincorporation, often with the isotope being distributed over many siteson the molecule.

The compounds, as disclosed herein ,can be prepared by methods known toone of skill in the art and routine modifications thereof, and/orfollowing procedures similar to those described in the Example sectionherein or schemes described in FIGS. 3-5 and routine modificationsthereof, and/or procedures found in WO 2014014841; WO 2013185112; WO2012170061; WO 2011133956; WO 2011133751; WO 2011119984; WO 2010054138;WO 2010053471; US 201301162389 US 20120046330; US 201200159999 US20090131492, which are hereby incorporated in their entirety, andreferences cited therein and routine modifications thereof. Exemplarymethods for the synthesis of some deuterated tigemonam derivatives areillustrated in FIGS. 3-5 and these, for example, could be adapted by theskilled artisan to produce more complex deuterated derivatives, byexample using various deuterated side chains when coupling to the lactamcore.

Compositions and Methods of Administration

The compositions provided herein contain therapeutically effectiveamounts of one or more of the compounds provided herein that are usefulin the prevention, treatment, or amelioration of one or more of thesymptoms of diseases or disorders described herein and a vehicle.Vehicles suitable for administration of the compounds provided hereininclude any such carriers known to those skilled in the art to besuitable for the particular mode of administration. In addition, thecompounds may be formulated as the sole active ingredient in thecomposition or may be combined with other active ingredients.

The compositions contain one or more compounds provided herein. Thecompounds are, in some embodiments, formulated into suitablepreparations such as solutions, suspensions, tablets, dispersibletablets, pills, capsules, powders, sustained release formulations orelixirs, for oral administration or in sterile solutions or suspensionsfor parenteral administration, as well as topical administration,transdermal administration and oral inhalation via nebulizers,pressurized metered dose inhalers and dry powder inhalers. In someembodiments, the compounds described above are formulated intocompositions using techniques and procedures well known in the art (see,e.g., Ansel, Introduction to Pharmaceutical Dosage Forms, SeventhEdition (1999)).

In the compositions, effective concentrations of one or more compoundsor derivatives thereof is (are) mixed with a suitable vehicle. Thecompounds may be derivatized as the corresponding salts, esters, enolethers or esters, acetals, ketals, orthoesters, hemiacetals, hemiketals,acids, bases, solvates, ion-pairs, hydrates or prodrugs prior toformulation, as described above. The concentrations of the compounds inthe compositions are effective for delivery of an amount, uponadministration that treats, leads to prevention, or amelioration of oneor more of the symptoms of diseases or disorders described herein. Insome embodiments, the compositions are formulated for single dosageadministration. To formulate a composition, the weight fraction of acompound is dissolved, suspended, dispersed or otherwise mixed in aselected vehicle at an effective concentration such that the treatedcondition is relieved, prevented, or one or more symptoms areameliorated.

The active compound is included in the vehicle in an amount sufficientto exert a therapeutically useful effect in the absence of undesirableside effects on the patient treated. The therapeutically effectiveconcentration may be predicted empirically by testing the compounds inin vitro and in vivo systems well known to those of skill in the art andthen extrapolated therefrom for dosages for humans. Human doses are thentypically fine-tuned in clinical trials and titrated to response.

The concentration of active compound in the composition will depend onabsorption, inactivation and excretion rates of the active compound, thephysicochemical characteristics of the compound, the dosage schedule,and amount administered as well as other factors known to those of skillin the art. For example, the amount that is delivered is sufficient toameliorate one or more of the symptoms of diseases or disorders asdescribed herein.

In instances in which the compounds exhibit insufficient solubility,methods for solubilizing compounds may be used such as use of liposomes,prodrugs, complexation/chelation, nanoparticles, or emulsions ortertiary templating. Such methods are known to those of skill in thisart, and include, but are not limited to, using co-solvents, such asdimethylsulfoxide (DMSO), using surfactants or surface modifiers, suchas TWEEN®, complexing agents such as cyclodextrin or dissolution byenhanced ionization (i.e. dissolving in aqueous sodium bicarbonate).Derivatives of the compounds, such as prodrugs of the compounds may alsobe used in formulating effective compositions.

Upon mixing or addition of the compound(s), the resulting mixture may bea solution, suspension, emulsion or the like. The form of the resultingmixture depends upon a number of factors, including the intended mode ofadministration and the solubility of the compound in the selectedvehicle. The effective concentration is sufficient for ameliorating thesymptoms of the disease, disorder or condition treated and may beempirically determined.

The compositions are provided for administration to humans and animalsin indication appropriate dosage forms, such as dry powder inhalers(DPIs), pressurized metered dose inhalers (pMDIs), nebulizers, tablets,capsules, pills, sublingual tapes/bioerodible strips, tablets orcapsules, powders, granules, lozenges, lotions, salves, suppositories,fast melts, transdermal patches or other transdermal applicationdevices/preparations, sterile parenteral solutions or suspensions, andoral solutions or suspensions, and oil-water emulsions containingsuitable quantities of the compounds or derivatives thereof. Thetherapeutically active compounds and derivatives thereof are, in someembodiments, formulated and administered in unit-dosage forms ormultiple-dosage forms. Unit-dose forms as used herein refer tophysically discrete units suitable for human and animal subjects andpackaged individually as is known in the art. Each unit-dose contains apredetermined quantity of the therapeutically active compound sufficientto produce the desired therapeutic effect, in association with therequired vehicle. Examples of unit-dose forms include ampoules andsyringes and individually packaged tablets or capsules. Unit-dose formsmay be administered in fractions or multiples thereof. A multiple-doseform is a plurality of identical unit-dosage forms packaged in a singlecontainer to be administered in segregated unit-dose form. Examples ofmultiple-dose forms include vials, bottles of tablets or capsules orbottles of pints or gallons. Hence, multiple dose form is a multiple ofunit-doses which are not segregated in packaging.

Liquid compositions can, for example, be prepared by dissolving,dispersing, or otherwise mixing an active compound as defined above andoptional adjuvants in a vehicle, such as, for example, water, saline,aqueous dextrose, glycerol, glycols, ethanol, and the like, to therebyform a solution or suspension, colloidal dispersion, emulsion orliposomal formulation. If desired, the composition to be administeredmay also contain minor amounts of nontoxic auxiliary substances such aswetting agents, emulsifying agents, solubilizing agents, pH bufferingagents and the like, for example, acetate, sodium citrate, cyclodextrinderivatives, sorbitan monolaurate, triethanolamine sodium acetate,triethanolamine oleate, and other such agents.

Actual methods of preparing such dosage forms are known, or will beapparent, to those skilled in this art; for example, see Remington'sPharmaceutical Sciences, Mack Publishing Company, Easton, Pa., 15thEdition, 1975 or later editions thereof.

Dosage forms or compositions containing active ingredient in the rangeof 0.005% to 100% with the balance made up from vehicle or carrier maybe prepared. Methods for preparation of these compositions are known tothose skilled in the art. The contemplated compositions may contain0.001%-100% active ingredient, in one embodiment 0.1-95%, in anotherembodiment 0.4-10%.

In certain embodiments, the compositions are lactose-free compositionscontaining excipients that are well known in the art and are listed, forexample, in the U.S. Pharmacopeia (USP) 25-NF20 (2002). In general,lactose-free compositions contain active ingredients, a binder/filler,and a lubricant in compatible amounts. Particular lactose-free dosageforms contain active ingredients, microcrystalline cellulose,pre-gelatinized starch, and magnesium stearate.

Further provided are anhydrous compositions and dosage forms comprisingactive ingredients, since water can facilitate the degradation of somecompounds. For example, the addition of water (e.g., 5%) is widelyaccepted as a means of simulating long-term storage in order todetermine characteristics such as shelf-life or the stability offormulations over time. See, e.g., Jens T. Carstensen, Drug Stability:Principles & Practice, 2d. Ed., Marcel Dekker, NY, NY, 1995, pp. 379-80.In effect, water and heat accelerate the decomposition of somecompounds. Thus, the effect of water on a formulation can be of greatsignificance since moisture and/or humidity are commonly encounteredduring manufacture, handling, packaging, storage, shipment, and use offormulations.

Anhydrous compositions and dosage forms provided herein can be preparedusing anhydrous or low moisture containing ingredients and low moistureor low humidity conditions.

An anhydrous composition should be prepared and stored such that itsanhydrous nature is maintained. Accordingly, anhydrous compositions aregenerally packaged using materials known to prevent exposure to watersuch that they can be included in suitable formulary kits. Examples ofsuitable packaging include, but are not limited to, hermetically sealedfoils, plastics, unit dose containers (e.g., vials), blister packs, andstrip packs.

Oral dosage forms are either solid, gel or liquid. The solid dosageforms are tablets, capsules, granules, and bulk powders. Types of oraltablets include compressed, chewable lozenges and tablets which may beenteric-coated, sugar-coated or film-coated. Capsules may be hard orsoft gelatin capsules, while granules and powders may be provided innon-effervescent or effervescent form with the combination of otheringredients known to those skilled in the art.

In certain embodiments, the formulations are solid dosage forms such asfor example, capsules or tablets. The tablets, pills, capsules, trochesand the like can contain one or more of the following ingredients, orcompounds of a similar nature: a binder; a lubricant; a diluent; aglidant; a disintegrating agent; a coloring agent; a sweetening agent; aflavoring agent; a wetting agent; an enteric coating; a film coatingagent and modified release agent. Examples of binders includemicrocrystalline cellulose, methyl paraben, polyalkyleneoxides, gumtragacanth, glucose solution, acacia mucilage, gelatin solution,molasses, polyvinylpyrrolidine, povidone, crospovidones, sucrose andstarch and starch derivatives. Lubricants include talc, starch,magnesium/calcium stearate, lycopodium and stearic acid. Diluentsinclude, for example, lactose, sucrose, trehalose, lysine, leucine,lecithin, starch, kaolin, salt, mannitol and dicalcium phosphate.Glidants include, but are not limited to, colloidal silicon dioxide.Disintegrating agents include crosscarmellose sodium, sodium starchglycolate, alginic acid, corn starch, potato starch, bentonite,methylcellulose, agar and carboxymethylcellulose. Coloring agentsinclude, for example, any of the approved certified water soluble FD andC dyes, mixtures thereof; and water insoluble FD and C dyes suspended onalumina hydrate and advanced coloring or anti-forgery color/opalescentadditives known to those skilled in the art. Sweetening agents includesucrose, lactose, mannitol and artificial sweetening agents such assaccharin and any number of spray dried flavors. Flavoring agentsinclude natural flavors extracted from plants such as fruits andsynthetic blends of compounds which produce a pleasant sensation or maskunpleasant taste, such as, but not limited to peppermint and methylsalicylate. Wetting agents include propylene glycol monostearate,sorbitan monooleate, diethylene glycol monolaurate and polyoxyethylenelauryl ether. Enteric-coatings include fatty acids, fats, waxes,shellac, ammoniated shellac and cellulose acetate phthalates. Filmcoatings include hydroxyethylcellulose, sodium carboxymethylcellulose,polyethylene glycol 4000 and cellulose acetate phthalate. Modifiedrelease agents include polymers such as the Eudragit® series andcellulose esters.

The compound, or derivative thereof, can be provided in a compositionthat protects it from the acidic environment of the stomach. Forexample, the composition can be formulated in an enteric coating thatmaintains its integrity in the stomach and releases the active compoundin the intestine. The composition may also be formulated in combinationwith an antacid or other such ingredient.

When the dosage unit form is a capsule, it can contain, in addition tomaterial of the above type, a liquid carrier such as a fatty oil. Inaddition, dosage unit forms can contain various other materials whichmodify the physical form of the dosage unit, for example, coatings ofsugar and other enteric agents. The compounds can also be administeredas a component of an elixir, suspension, syrup, wafer, sprinkle, chewinggum or the like. A syrup may contain, in addition to the activecompounds, sucrose as a sweetening agent and certain preservatives, dyesand colorings and flavors.

The active materials can also be mixed with other active materials whichdo not impair the desired action, or with materials that supplement thedesired action, such as antacids, H₂ blockers, and diuretics. The activeingredient is a compound or derivative thereof as described herein.Higher concentrations, up to about 98% by weight of the activeingredient may be included.

In all embodiments, tablets and capsules formulations may be coated asknown by those of skill in the art in order to modify or sustaindissolution of the active ingredient. Thus, for example, they may becoated with a conventional enterically digestible coating, such asphenylsalicylate, waxes and cellulose acetate phthalate.

Liquid oral dosage forms include aqueous solutions, emulsions,suspensions, solutions and/or suspensions reconstituted fromnon-effervescent granules and effervescent preparations reconstitutedfrom effervescent granules. Aqueous solutions include, for example,elixirs and syrups. Emulsions are either oil-in-water or water-in-oil.

Elixirs are clear, sweetened, hydroalcoholic preparations. Vehicles usedin elixirs include solvents. Syrups are concentrated aqueous solutionsof a sugar, for example, sucrose, and may contain a preservative. Anemulsion is a two-phase system in which one liquid is dispersed in theform of small globules throughout another liquid. Carriers used inemulsions are non-aqueous liquids, emulsifying agents and preservatives.Suspensions use suspending agents and preservatives. Acceptablesubstances used in non-effervescent granules, to be reconstituted into aliquid oral dosage form, include diluents, sweeteners and wettingagents. Acceptable substances used in effervescent granules, to bereconstituted into a liquid oral dosage form, include organic acids anda source of carbon dioxide. Coloring and flavoring agents are used inall of the above dosage forms.

Solvents include glycerin, sorbitol, ethyl alcohol and syrup. Examplesof preservatives include glycerin, methyl and propylparaben, benzoicacid, sodium benzoate and alcohol. Examples of non-aqueous liquidsutilized in emulsions include mineral oil and cottonseed oil. Examplesof emulsifying agents include gelatin, acacia, tragacanth, bentonite,and surfactants such as polyoxyethylene sorbitan monooleate. Suspendingagents include sodium carboxymethylcellulose, pectin, tragacanth, Veegumand acacia. Sweetening agents include sucrose, syrups, glycerin andartificial sweetening agents such as saccharin. Wetting agents includepropylene glycol monostearate, sorbitan monooleate, diethylene glycolmonolaurate and polyoxyethylene lauryl ether. Organic acids includecitric and tartaric acid. Sources of carbon dioxide include sodiumbicarbonate and sodium carbonate. Coloring agents include any of theapproved certified water soluble FD and C dyes, and mixtures thereof.Flavoring agents include natural flavors extracted from plants suchfruits, and synthetic blends of compounds which produce a pleasant tastesensation.

For a solid dosage form, the solution or suspension, in for example,propylene carbonate, vegetable oils or triglycerides, is in someembodiments encapsulated in a gelatin capsule. Such solutions, and thepreparation and encapsulation thereof, are disclosed in U.S. Pat. Nos.4,328,245; 4,409,239; and 4,410,545. For a liquid dosage form, thesolution, e.g., for example, in a polyethylene glycol, may be dilutedwith a sufficient quantity of a liquid vehicle, e.g., water, to beeasily measured for administration.

Alternatively, liquid or semi-solid oral formulations may be prepared bydissolving or dispersing the active compound or salt in vegetable oils,glycols, triglycerides, propylene glycol esters (e.g., propylenecarbonate) and other such carriers, and encapsulating these solutions orsuspensions in hard or soft gelatin capsule shells. Other usefulformulations include those set forth in U.S. Pat. Nos. RE28,819 and4,358,603. Briefly, such formulations include, but are not limited to,those containing a compound provided herein, a dialkylated mono- orpolyalkylene glycol, including, but not limited to, 1,2-dimethoxyethane,diglyme, triglyme, tetraglyme, polyethylene glycol-350-dimethyl ether,polyethylene glycol-550-dimethyl ether, polyethylene glycol-750-dimethylether wherein 350, 550 and 750 refer to the approximate averagemolecular weight of the polyethylene glycol, and one or moreantioxidants, such as butylated hydroxytoluene (BHT), butylatedhydroxyanisole (BHA), propyl gallate, vitamin E, hydroquinone,hydroxycoumarins, ethanolamine, lecithin, cephalin, ascorbic acid, malicacid, sorbitol, phosphoric acid, thiodipropionic acid and its esters,and dithiocarbamates.

Other formulations include, but are not limited to, aqueous alcoholicsolutions including an acetal. Alcohols used in these formulations areany water-miscible solvents having one or more hydroxyl groups,including, but not limited to, propylene glycol and ethanol. Acetalsinclude, but are not limited to, di(lower alkyl) acetals of lower alkylaldehydes such as acetaldehyde diethyl acetal.

Parenteral administration, in some embodiments characterized byinjection, either subcutaneously, intramuscularly or intravenously isalso contemplated herein. Injectables can be prepared in conventionalforms, either as liquid solutions or suspensions, solid forms suitablefor solution or suspension in liquid prior to injection, or asemulsions. The injectables, solutions and emulsions also contain one ormore excipients. Suitable excipients are, for example, water, saline,dextrose, glycerol or ethanol. In addition, if desired, the compositionsto be administered may also contain minor amounts of non-toxic auxiliarysubstances such as wetting or emulsifying agents, pH buffering agents,stabilizers, solubility enhancers, and other such agents, such as forexample, sodium acetate, sorbitan monolaurate, triethanolamine oleateand cyclodextrins.

Implantation of a slow-release or sustained-release system, such that aconstant level of dosage is maintained (see, e.g., U.S. Pat. No.3,710,795) is also contemplated herein. Briefly, a compound providedherein is dispersed in a solid inner matrix, e.g.,polymethylmethacrylate, polybutylmethacrylate, plasticized orunplasticized polyvinylchloride, plasticized nylon, plasticizedpolyethyleneterephthalate, natural rubber, polyisoprene,polyisobutylene, polybutadiene, polyethylene, ethylene-vinylacetatecopolymers, silicone rubbers, polydimethylsiloxanes, silicone carbonatecopolymers, hydrophilic polymers such as hydrogels of esters of acrylicand methacrylic acid, collagen, cross-linked polyvinylalcohol andcross-linked partially hydrolyzed polyvinyl acetate, that is surroundedby an outer polymeric membrane, e.g., polyethylene, polypropylene,ethylene/propylene copolymers, ethylene/ethyl acrylate copolymers,ethylene/vinylacetate copolymers, silicone rubbers, polydimethylsiloxanes, neoprene rubber, chlorinated polyethylene, polyvinylchloride,vinylchloride copolymers with vinyl acetate, vinylidene chloride,ethylene and propylene, ionomer polyethylene terephthalate, butyl rubberepichlorohydrin rubbers, ethylene/vinyl alcohol copolymer,ethylene/vinyl acetate/vinyl alcohol terpolymer, andethylene/vinyloxyethanol copolymer, that is insoluble in body fluids.The compound diffuses through the outer polymeric membrane in a releaserate controlling step. The percentage of active compound contained insuch parenteral compositions is highly dependent on the specific naturethereof, as well as the activity of the compound and the needs of thesubject.

Parenteral administration of the compositions includes intravenous,subcutaneous and intramuscular administrations. Preparations forparenteral administration include sterile solutions ready for injection,sterile dry soluble products, such as lyophilized powders, ready to becombined with a solvent just prior to use, including hypodermic tablets,sterile suspensions ready for injection, sterile dry insoluble productsready to be combined with a vehicle just prior to use and sterileemulsions. The solutions may be either aqueous or nonaqueous.

If administered intravenously, suitable carriers include physiologicalsaline or phosphate buffered saline (PBS), and solutions containingthickening and solubilizing agents, such as glucose, polyethyleneglycol, and polypropylene glycol and mixtures thereof.

Vehicles used in parenteral preparations include aqueous vehicles,nonaqueous vehicles, antimicrobial agents, isotonic agents, buffers,antioxidants, local anesthetics, suspending and dispersing agents,emulsifying agents, sequestering or chelating agents and othersubstances.

Examples of aqueous vehicles include Sodium Chloride Injection, RingersInjection, Isotonic Dextrose Injection, Sterile Water Injection,Dextrose and Lactated Ringers Injection. Nonaqueous parenteral vehiclesinclude fixed oils of vegetable origin, cottonseed oil, corn oil, sesameoil and peanut oil. Antimicrobial agents in bacteriostatic orfungistatic concentrations must be added to parenteral preparationspackaged in multiple-dose containers which include phenols or cresols,mercurials, benzyl alcohol, chlorobutanol, methyl and propylp-hydroxybenzoic acid esters, thimerosal, benzalkonium chloride andbenzethonium chloride. Isotonic agents include sodium chloride anddextrose. Buffers include phosphate and citrate. Antioxidants includesodium bisulfate. Local anesthetics include procaine hydrochloride.Suspending and dispersing agents include sodium carboxymethylcellulose,hydroxypropyl methylcellulose and polyvinylpyrrolidone. Emulsifyingagents include Polysorbate 80 (Tween® 80). A sequestering or chelatingagent of metal ions includes EDTA. Carriers also include ethyl alcohol,polyethylene glycol and propylene glycol for water miscible vehicles;and sodium hydroxide, hydrochloric acid, citric acid or lactic acid forpH adjustment.

The concentration of compound is adjusted so that an injection providesan effective amount to produce the desired pharmacological effect. Theexact dose depends on the age, weight, body surface area and conditionof the patient or animal as is known in the art.

The unit-dose parenteral preparations are packaged in an ampoule, a vialor a syringe with a needle. All preparations for parenteraladministration must be sterile, as is known and practiced in the art.

Illustratively, intravenous or intraarterial infusion of a sterileaqueous solution containing an active compound is an effective mode ofadministration. Another embodiment is a sterile aqueous or oily solutionor suspension containing an active material injected as necessary toproduce the desired pharmacological effect.

Injectables are designed for local and systemic administration. In someembodiments, a therapeutically effective dosage is formulated to containa concentration of at least about 0.01% w/w up to about 90% w/w or more,in certain embodiments more than 0.1% w/w of the active compound to thetreated tissue(s).

The compound may be suspended in micronized or other suitable form ormay be derivatized to produce a more soluble active product or toproduce a prodrug. The form of the resulting mixture depends upon anumber of factors, including the intended mode of administration and thesolubility of the compound in the selected carrier or vehicle. Theeffective concentration is sufficient for ameliorating the symptoms ofthe condition and may be empirically determined.

Active ingredients provided herein can be administered by controlledrelease means or by delivery devices that are well known to those ofordinary skill in the art. Examples include, but are not limited to,those described in U.S. Pat. Nos. 3,845,770; 3,916,899; 3,536,809;3,598,123; 4,008,719; 5,674,533; 5,059,595; 5,591,767; 5,120,548;5,073,543; 5,639,476; 5,354,556; 5,639,480; 5,733,566; 5,739,108;5,891,474; 5,922,356; 5,972,891; 5,980,945; 5,993,855; 6,045,830;6,087,324; 6,113,943; 6,197,350; 6,248,363; 6,264,970; 6,267,981;6,376,461; 6,419,961; 6,589,548; 6,613,358; 6,699,500 and 6,740,634.Such dosage forms can be used to provide slow or controlled-release ofone or more active ingredients using, for example, hydroxypropylmethylcellulose, other polymer matrices, gels, permeable membranes, osmoticsystems, multilayer coatings, microparticles, liposomes, microspheres,or a combination thereof to provide the desired release profile invarying proportions. Suitable controlled-release formulations known tothose of ordinary skill in the art, including those described herein,can be readily selected for use with the active ingredients providedherein.

All controlled-release products have a common goal of improving drugtherapy over that achieved by their non-controlled counterparts.Ideally, the use of an optimally designed controlled-release preparationin medical treatment is characterized by a minimum of drug substancebeing employed to cure or control the condition in a minimum amount oftime. Advantages of controlled-release formulations include extendedactivity of the drug, reduced dosage frequency, and increased patientcompliance. In addition, controlled-release formulations can be used toaffect the time of onset of action or other characteristics, such asblood levels of the drug, and can thus affect the occurrence of side(e.g., adverse) effects.

Most controlled-release formulations are designed to initially releasean amount of drug (active ingredient) that promptly produces the desiredtherapeutic effect, and gradually and continually release of otheramounts of drug to maintain this level of therapeutic or prophylacticeffect over an extended period of time. In order to maintain thisconstant level of drug in the body, the drug must be released from thedosage form at a rate that will replace the amount of drug beingmetabolized and excreted from the body. Controlled-release of an activeingredient can be stimulated by various conditions including, but notlimited to, pH, temperature, enzymes, water, or other physiologicalconditions or compounds.

In certain embodiments, the agent may be administered using intravenousinfusion, an implantable osmotic pump, a transdermal patch, liposomes,or other modes of administration. In some embodiments, a pump may beused (see, Sefton, CRC Crit. Ref. Biomed. Eng. 14:201 (1987); Buchwaldet al., Surgery 88:507 (1980); Saudek et al., N. Engl. J. Med. 321:574(1989)). In other embodiments, polymeric materials can be used. In otherembodiments, a controlled release system can be placed in proximity ofthe therapeutic target, i.e., thus requiring only a fraction of thesystemic dose (see, e.g., Goodson, Medical Applications of ControlledRelease, vol. 2, pp. 115-138 (1984)). In some embodiments, a controlledrelease device is introduced into a subject in proximity of the site ofinappropriate immune activation or a tumor. Other controlled releasesystems are discussed in the review by Langer (Science 249:1527-1533(1990)). The active ingredient can be dispersed in a solid inner matrix,e.g., polymethylmethacrylate, polybutylmethacrylate, plasticized orunplasticized polyvinylchloride, plasticized nylon, plasticizedpolyethyleneterephthalate, natural rubber, polyisoprene,polyisobutylene, polybutadiene, polyethylene, ethylene-vinylacetatecopolymers, silicone rubbers, polydimethylsiloxanes, silicone carbonatecopolymers, hydrophilic polymers such as hydrogels of esters of acrylicand methacrylic acid, collagen, cross-linked polyvinylalcohol andcross-linked partially hydrolyzed polyvinyl acetate, that is surroundedby an outer polymeric membrane, e.g., polyethylene, polypropylene,ethylene/propylene copolymers, ethylene/ethyl acrylate copolymers,ethylene/vinylacetate copolymers, silicone rubbers, polydimethylsiloxanes, neoprene rubber, chlorinated polyethylene, polyvinylchloride,vinylchloride copolymers with vinyl acetate, vinylidene chloride,ethylene and propylene, ionomer polyethylene terephthalate, butyl rubberepichlorohydrin rubbers, ethylene/vinyl alcohol copolymer,ethylene/vinyl acetate/vinyl alcohol terpolymer, andethylene/vinyloxyethanol copolymer, that is insoluble in body fluids.The active ingredient then diffuses through the outer polymeric membranein a release rate controlling step. The percentage of active ingredientcontained in such parenteral compositions is highly dependent on thespecific nature thereof, as well as the needs of the subject.

Of interest, herein are also lyophilized powders, which can bereconstituted for administration as solutions, emulsions and othermixtures. They may also be reconstituted and formulated as solids orgels.

The sterile, lyophilized powder is prepared by dissolving a compoundprovided herein, or a derivative thereof, in a suitable solvent. Thesolvent may contain an excipient which improves the stability or otherpharmacological component of the powder or reconstituted solution,prepared from the powder.

Excipients that may be used include, but are not limited to, anantioxidant, a buffer and a bulking agent. In some embodiments, theexcipient is selected from dextrose, sorbitol, fructose, corn syrup,xylitol, glycerin, glucose, sucrose and other suitable agents. Thesolvent may contain a buffer, such as citrate, sodium or potassiumphosphate or other such buffer known to those of skill in the art at, atabout neutral pH. Subsequent sterile filtration of the solution followedby lyophilization under standard conditions known to those of skill inthe art provides the desired formulation. In some embodiments, theresulting solution will be apportioned into vials for lyophilization.Each vial will contain a single dosage or multiple dosages of thecompound. The lyophilized powder can be stored under appropriateconditions, such as at about 4° C. to room temperature.

Reconstitution of this lyophilized powder with water for injectionprovides a formulation for use in parenteral administration. Forreconstitution, the lyophilized powder is added to sterile water orother suitable carriers. The precise amount depends upon the selectedcompound. Such amount can be empirically determined.

Topical mixtures are prepared as described for the local and systemicadministration. The resulting mixture may be a solution, suspension,emulsions or the like and are formulated as creams, gels, ointments,emulsions, solutions, elixirs, lotions, suspensions, tinctures, pastes,foams, aerosols, irrigations, sprays, suppositories, bandages, dermalpatches or any other formulations suitable for topical administration.

The compounds or derivatives thereof may be formulated as aerosols fortopical application, such as by inhalation (see, e.g., U.S. Pat. Nos.4,044,126, 4,414,209, and 4,364,923, which describe aerosols fordelivery of a steroid useful for treatment of inflammatory diseases,particularly asthma). These formulations for administration to therespiratory tract can be in the form of an aerosol or solution for anebulizer, or as a microfine powder for insufflation, alone or incombination with an inert carrier such as lactose. In such a case, theparticles of the formulation will, in some embodiments, have mass mediangeometric diameters of less than 5 microns, in other embodiments lessthan 10 microns.

Oral inhalation formulations of the compounds or derivatives suitablefor inhalation include metered dose inhalers, dry powder inhalers andliquid preparations for administration from a nebulizer or metered doseliquid dispensing system. For both metered dose inhalers and dry powderinhalers, a crystalline form of the compounds or derivatives is thepreferred physical form of the drug to confer longer product stability.

In addition to particle size reduction methods known to those skilled inthe art, crystalline particles of the compounds or derivatives can begenerated using supercritical fluid processing which offers significantadvantages in the production of such particles for inhalation deliveryby producing respirable particles of the desired size in a single step.(e.g., International Publication No. WO2005/025506). A controlledparticle size for the microcrystals can be selected to ensure that asignificant fraction of the compounds or derivatives is deposited in thelung. In some embodiments, these particles have a mass medianaerodynamic diameter of about 0.1 to about 10 microns, in otherembodiments, about 1 to about 5 microns and still other embodiments,about 1.2 to about 3 microns.

Inert and non-flammable HFA propellants are selected from HFA 134a(1,1,1,2-tetrafluoroethane) and HFA 227e(1,1,1,2,3,3,3-heptafluoropropane) and provided either alone or as aratio to match the density of crystal particles of the compounds orderivatives. A ratio is also selected to ensure that the productsuspension avoids detrimental sedimentation or cream (which canprecipitate irreversible agglomeration) and instead promote a looselyflocculated system, which is easily dispersed when shaken. Looselyfluctuated systems are well regarded to provide optimal stability forpMDI canisters. As a result of the formulation's properties, theformulation contained no ethanol and no surfactants/stabilizing agents.

The compounds may be formulated for local or topical application, suchas for topical application to the skin and mucous membranes, such as inthe eye, in the form of gels, creams, and lotions and for application tothe eye or for intracisternal or intraspinal application. Topicaladministration is contemplated for transdermal delivery and also foradministration to the eyes or mucosa, or for inhalation therapies. Nasalsolutions of the active compound alone or in combination with otherexcipients can also be administered.

For nasal administration, the preparation may contain an esterifiedphosphonate compound dissolved or suspended in a liquid carrier, inparticular, an aqueous carrier, for aerosol application. The carrier maycontain solubilizing or suspending agents such as propylene glycol,surfactants, absorption enhancers such as lecithin or cyclodextrin, orpreservatives.

Solutions, particularly those intended for ophthalmic use, may beformulated as 0.01%-10% isotonic solutions, pH about 5-7.4, withappropriate salts.

Other routes of administration, such as transdermal patches, includingiontophoretic and electrophoretic devices, and rectal administration,are also contemplated herein.

Transdermal patches, including iontophoretic and electrophoreticdevices, are well known to those of skill in the art. For example, suchpatches are disclosed in U.S. Pat. Nos. 6,267,983, 6,261,595, 6,256,533,6,167,301, 6,024,975, 6,010715, 5,985,317, 5,983,134, 5,948,433 and5,860,957.

For example, dosage forms for rectal administration are rectalsuppositories, capsules and tablets for systemic effect. Rectalsuppositories are used herein mean solid bodies for insertion into therectum which melt or soften at body temperature releasing one or morepharmacologically or therapeutically active ingredients. Substancesutilized in rectal suppositories are bases or vehicles and agents toraise the melting point. Examples of bases include cocoa butter(theobroma oil), glycerin-gelatin, carbowax (polyoxyethylene glycol) andappropriate mixtures of mono-, di- and triglycerides of fatty acids.Combinations of the various bases may be used. Agents to raise themelting point of suppositories include spermaceti and wax. Rectalsuppositories may be prepared either by the compressed method or bymolding. The weight of a rectal suppository, in one embodiment, is about2 to 3 gm. Tablets and capsules for rectal administration aremanufactured using the same substance and by the same methods as forformulations for oral administration.

The compounds provided herein, or derivatives thereof, may also beformulated to be targeted to a particular tissue, receptor, or otherarea of the body of the subject to be treated. Many such targetingmethods are well known to those of skill in the art. All such targetingmethods are contemplated herein for use in the instant compositions. Fornon-limiting examples of targeting methods, see, e.g., U.S. Pat. Nos.6,316,652, 6,274,552, 6,271,359, 6,253,872, 6,139,865, 6,131,570,6,120,751, 6,071,495, 6,060,082, 6,048,736, 6,039,975, 6,004,534,5,985,307, 5,972,366, 5,900,252, 5,840,674, 5,759,542 and 5,709,874.

In some embodiments, liposomal suspensions, including tissue-targetedliposomes, such as tumor-targeted liposomes, may also be suitable ascarriers. These may be prepared according to methods known to thoseskilled in the art. For example, liposome formulations may be preparedas described in U.S. Pat. No. 4,522,811. Briefly, liposomes such asmultilamellar vesicles (MLV's) may be formed by drying down phosphatidylcholine and phosphatidyl serine (7:3 molar ratio) on the inside of aflask. A solution of a compound provided herein in phosphate bufferedsaline lacking divalent cations (PBS) is added and the flask shakenuntil the lipid film is dispersed. The resulting vesicles are washed toremove unencapsulated compound, pelleted by centrifugation, and thenresuspended in PBS.

The compounds or derivatives may be packaged as articles of manufacturecontaining packaging material, a compound or derivative thereof providedherein, which is effective for treatment, prevention or amelioration ofone or more symptoms of the diseases or disorders, supra, within thepackaging material, and a label that indicates that the compound orcomposition or derivative thereof, is used for the treatment, preventionor amelioration of one or more symptoms of the diseases or disorders,supra.

The articles of manufacture provided herein contain packaging materials.Packaging materials for use in packaging products are well known tothose of skill in the art. See, e.g., U.S. Pat. Nos. 5,323,907,5,052,558 and 5,033,252. Examples of packaging materials include, butare not limited to, blister packs, bottles, tubes, inhalers, pumps,bags, vials, containers, syringes, bottles, and any packaging materialsuitable for a selected formulation and intended mode of administrationand treatment. A wide array of formulations of the compounds andcompositions provided herein are contemplated as are a variety oftreatments for any disease or disorder described herein.

Dosages

For use to treat or prevent infectious disease, the compounds describedherein, or pharmaceutical compositions thereof, are administered orapplied in a therapeutically effective amount. In human therapeutics,the physician will determine the dosage regimen that is most appropriateaccording to a preventive or curative treatment and according to theage, weight, stage of the disease and other factors specific to thesubject to be treated. The amount of active ingredient in theformulations provided herein, which will be effective in the preventionor treatment of an infectious disease will vary with the nature andseverity of the disease or condition, and the route by which the activeingredient is administered. The frequency and dosage will also varyaccording to factors specific for each subject depending on the specifictherapy (e.g., therapeutic or prophylactic agents) administered, theseverity of the infection, the route of administration, as well as age,body, weight, response, and the past medical history of the subject.

Exemplary doses of a formulation include milligram or microgram amountsof the active compound per kilogram of subject (e.g., from about 1microgram per kilogram to about 50 milligrams per kilogram, from about10 micrograms per kilogram to about 30 milligrams per kilogram, fromabout 100 micrograms per kilogram to about 10 milligrams per kilogram,or from about 100 micrograms per kilogram to about 5 milligrams perkilogram).

In some embodiments, a therapeutically effective dosage should produce aserum concentration of active ingredient of from about 0.001 ng/ml toabout 50-200 μg/ml. The compositions, in other embodiments, shouldprovide a dosage of from about 0.0001 mg to about 70 mg of compound perkilogram of body weight per day. Dosage unit forms are prepared toprovide from about 0.01 mg, 0.1 mg or 1 mg to about 500 mg, 1000 mg or5000 mg, and in some embodiments from about 10 mg to about 500 mg of theactive ingredient or a combination of essential ingredients per dosageunit form.

The active ingredient may be administered at once, or may be dividedinto a number of smaller doses to be administered at intervals of time.It is understood that the precise dosage and duration of treatment is afunction of the disease being treated and may be determined empiricallyusing known testing protocols or by extrapolation from in vivo or invitro test data or subsequent clinical testing. It is to be noted thatconcentrations and dosage values may also vary with the severity of thecondition to be alleviated. It is to be further understood that for anyparticular subject, specific dosage regimens should be adjusted overtime according to the individual need and the professional judgment ofthe person administering or supervising the administration of thecompositions and that the concentration ranges set forth herein areexemplary only and are not intended to limit the scope or practice ofthe claimed compositions.

It may be necessary to use dosages of the active ingredient outside theranges disclosed herein in some cases, as will be apparent to those ofordinary skill in the art. Furthermore, it is noted that the clinicianor treating physician will know how and when to interrupt, adjust, orterminate therapy in conjunction with subject response.

For systemic administration, a therapeutically effective dose can beestimated initially from in vitro assays. For example, a dose can beformulated in animal models to achieve a circulating concentration rangethat includes the IC₅₀ as determined in cell culture (i.e., theconcentration of test compound that is lethal to 50% of a cell culture),the MIC as determined in cell culture (i.e., the minimal inhibitoryconcentration for growth) or the IC₁₀₀ as determined in cell culture(i.e., the concentration of antimicrobial sulfonamide derivative that islethal to 100% of a cell culture). Such information can be used to moreaccurately determine useful doses in humans.

Initial dosages can also be estimated from in vivo data (e.g., animalmodels) using techniques that are well known in the art. One of ordinaryskill in the art can readily optimize administration to humans based onanimal data.

Alternatively, initial dosages can be determined from the dosagesadministered of known antimicrobial agents by comparing the IC₅₀, MICand/or I₁₀₀ of the specific compound disclosed herein with that of aknown antimicrobial agent, and adjusting the initial dosagesaccordingly. The optimal dosage may be obtained from these initialvalues by routine optimization

In cases of local administration or selective uptake, the effectivelocal concentration compound used may not be related to plasmaconcentration. One of skill in the art will be able to optimizetherapeutically effective local dosages without undue experimentation.

Ideally, a therapeutically effective dose of the compounds describedherein will provide therapeutic benefit without causing substantialtoxicity. Toxicity of compounds can be determined using standardpharmaceutical procedures in cell cultures or experimental animals,e.g., by determining the LD₅₀ (the dose lethal to 50% of the population)or the LD₁₀₀ (the dose lethal to 100% of the population). The dose ratiobetween toxic and therapeutic effect is the therapeutic index. Compoundswhich exhibit high therapeutic indices are preferred. The data obtainedfrom these cell culture assays and animal studies can be used informulating a dosage range that is not toxic for use in subjects. Thedosage of the compounds described herein lies preferably within a rangeof circulating concentrations that include the effective dose withlittle or no toxicity. The dosage may vary within this range dependingupon the dosage form employed and the route of administration utilized.The exact formulation, route of administration and dosage can be chosenby the individual physician in view of the patient's condition (See,e.g., Fingl et al., 1975, In: The Pharmacological Basis of Therapeutics,Ch.1, p.1).

The therapy may be repeated intermittently while infections aredetectable, or even when they are not detectable. In certainembodiments, administration of the same formulation provided herein maybe repeated and the administrations may be separated by at least 1 day,2 days, 3 days, 5 days, 10 days, 15 days, 30 days, 45 days, 2 months, 75days, 3 months, or 6 months.

Methods of Use of the Compounds and Compositions

The compounds and compositions described herein can be used in a widevariety of applications to treat or prevent infectious diseases in asubject. The methods generally involve administering therapeuticallyeffective amounts of deuterated O-sulfated beta-lactam hydroxamic acidsand/or deuterated N-sulfated beta-lactams, or a pharmaceuticalcomposition thereof to the subject.

In some embodiments, the infectious disease is a bacterial infection. Inother embodiments, the bacterial infection is an infection with aGram-negative bacterium. In still other embodiments, the Gram-negativebacteria is of one of the following genera: Acinetobacter, Aeromonas,Bacteroides, Burkholderia, Citrobacter, Enterobacter, Escherichia,Fusobacterium, Haemophilus, Klebsiella, Moraxella, Morganella,Mycoplasma, Neisseria, Pantoea, Pasteurella, Plesiomonas, Porphyromonas,Prevotella, Proteus, Providencia, Pseudomonas, Salmonella, Serratia,Shigella, Spirillum, Stenotrophomonas, Streptobacillus, Treponema, orYersinia. In still other embodiments, the Gram negative bacteria of isone of the following species: Acinetobacter baumannii, Aeromonashydrophila, Arizona hinshawii, Bacteroides fragilis, Branhamellacatarrhalis, Burkholderia cepacia, Citrobacter diversus, Citrobacterfreundii, Enterobacter aerogenes, Enterobacter cloacae, Escherichiacoli, Fusobacterium nucleatum, Haemophilus influenzae, Haemophilusparainfluenzae, Klebsiella oxytoca, Klebsiella pneumoniae, Moraxellacatarrhalis, Morganella morganii, Neisseria gonorrhoeae, Neisseriameningitidis, Pantoea agglomerans, Pasteurella multocida, Plesiomonasshigelloides, Prevotella melaninogenica, Proteus mirabilis, Proteusrettgeri, Proteus vulgaris, Pseudomonas aeruginosa, Pseudomonasdiminuta, Pseudomonas fluorescens, Pseudomonas stutzeri, Salmonellaenterica, Salmonella enteritidis, Salmonella typhi, Serratia marcescens,Spirillum minus, Stenotrophomonas maltophilia, Streptobacillusmoniliformis, Treponema pallidum, or Yersinia enterocolitica.

The compounds and compositions described herein may be used treat orprevent various diseases caused by the above bacteria. These include,but are not limited to, venereal disease, pneumonia, complicated urinarytract infections, urinary tract infections, complicated intra-abdominalinfections and intra-abdominal infections. The compounds andcompositions described herein may also be used kill bacteria describedabove. In some embodiments, the compounds and/or compositions areeffective against gram positive and anaerobic bacteria.

Additionally, the development of antibiotic resistance continues to growas a problem facing patients and clinicians. Accordingly, the US Foodand Drug Administration has identified the following pathogens aspresenting a potentially serious threat to public health: Acinetobacterspecies, Aspergillus species, Burkholderia cepacia complex,Campylobacter species, Candida species, Clostridium difficile,Coccidioides species, Cryptococcus species, Enterobacteriaceae (e.g.,Klebsiella pneumoniae), Enterococcus species, Helicobacter pylori,Mycobacterium tuberculosis complex, Neisseria gonorrhoeae, N.meningitidis, non-tuberculous mycobacteria species, Pseudomonas species,Staphylococcus aureus, Streptococcus agalactiae, S. pneumoniae, S.pyogenes, and Vibrio cholerae. The FDA has designated these organisms“qualifying pathogens” for purposes of the Generating AntibioticIncentives Now (GAIN) Act, intended to encourage development of newantibacterial and antifungal drugs for the treatment of serious or lifethreatening infections. The methods, kits, etc. disclosed herein areuseful for the treatment of diseases, infections, etc. caused by many ofthese organisms as well.

Combination Therapy

The compounds and compositions disclosed herein may also be used incombination with one or more other active ingredients. In certainembodiments, the compounds may be administered in combination, orsequentially, with another therapeutic agent. Such other therapeuticagents include those known for treatment, prevention, or amelioration ofinfectious disease. In some embodiments, the compounds andpharmaceutical compositions disclosed herein are administered withβ-lactamase inhibitors and/or carbapenemase inhibitors or pharmaceuticalcompositions thereof. Exemplary β-lactamase inhibitors and/orcarbapenemase inhibitors are well known to those of skill in the art andinclude, for example, clavulanic acid, sulbactam, avibactam, tazobactam,relebactam, vaborbactam, ETX 2514, RG6068 (i.e., OP0565) (Livermore etal., J AntiMicrob Chemother 2015, 70: 3032) and RPX7009 (Hecker et al.,J Med Chem 2015 58: 3682-3692). In other embodiments, the compounds andpharmaceutical compositions disclosed herein are administered withlindamycin, erythromycin, metronidazole, penicillins, or vancomycin orpharmaceutical compositions thereof. In still other embodiments, thecompounds and pharmaceutical compositions disclosed herein areadministered with administered with β-lactamase inhibitors and/orcarbapenemase inhibitors or pharmaceutical compositions thereof andlindamycin, erythromycin, metronidazole, penicillins, or vancomycin orpharmaceutical compositions thereof.

It should be understood that any suitable combination of the compoundsand pharmaceutical compositions provided herein with one or more of theabove therapeutic agents and optionally one or more furtherpharmacologically active substances are considered to be within thescope of the present disclosure. In some embodiments, the compounds andpharmaceutical compositions provided herein are administered prior to orsubsequent to the one or more additional active ingredients.

The following examples are provided for illustrative purposes only andare not intended to limit the scope of the invention.

EXAMPLES Example 1 d₆-(R)-tert-butyl(1,3-dihydroxy-3-methylbutan-2-yl)carbamate

A 1 M solution of d₃-Methylmagnesium iodide (200 ml, 200.0 mmol, 5.9equiv.) in diethyl ether was added dropwise to a cooled solution ofN-(tert-butoxycarbonyl)-D-serine (7.44 g, 33.9 mmol, 1.0 equiv.) indiethyl ether (12 ml) at −78° C. within 10 min. The reaction mixture wasallowed to reach r.t. and stirred at r.t. for 12 h at which point TLCand crude ¹H NMR indicated the reaction was complete. The reactionmixture was poured into a saturated aqueous ammonium chloride solution(250 mL). The organic layer was separated and the aqueous layer wasextracted with ethyl acetate (3×150 mL). The combined organic layerswere dried over anhydrous sodium sulfate, filtered and concentrated. Theresulting material was used directly without purification (7 g, 92%).¹HNMR (300 MHz, CDCl₃), δ 5.36 (m, 1H), 4.05-4.01 (m, 1H), 3.84-3.78 (m,1H), 3.48-3.45 (m, 1H), 2.53-2.44 (m, 2H), 1.45 (s, 9H).

Example 2 Synthesis ofd₆-(S)-2-((tert-butoxycarbonyl)amino)-3-hydroxy-3-methylbutanoic acid

TEMPO (0.49 g, 3.1 mmol, 0.1 equiv.) was added to a mixture ofd₆-(R)-tert-butyl (1,3-dihydroxy-3-methylbutan-2-yl)carbamate (7 g, 31.1mmol, 1.0 equiv.) in acetonitrile (155 mL) and sodium phosphate buffer(126 mL, 0.7 M). The resulting mixture was heated to 35° C. and thentreated by simultaneous addition of 80.0% sodium chlorite (9.1 g, 80.8mmol, 2.6 equiv.) solution in water (25 mL) and 4 drops of a very dilutesodium hypochlorite (has to be freshly made or at least within 3 days, 3mL of commercial solution in 100 mL of water), stirred at 35° C.overnight, cooled to r.t., treated with citric acid (6.4 g, 33.3 mmol,1.1 equiv.) (pH 3), saturated with sodium chloride and extracted withethyl acetate (280 ml) (3×). The organic extracts were combined andsodium carbonate solution was added (2M (314 ml, 627.7 mmol, 20.2equiv.)) and stirred overnight. After separation, the aqueous layer wasextracted with ethyl acetate again. The aqueous layer was cooled to 0°C., the pH was adjusted to 3.0 using 4 M solution of H₃PO₄ (313 mL) andthe solution was saturated with sodium chloride. The resulting mixturewas extracted with ethyl acetate (280 ml) (3×), the organic phases werecombined, dried, filtered and concentrated under reduced pressure togive the title compound as a white solid (5.02 g, 68%). ¹HNMR (300 MHz,DMSO-d6), δ 6.52 (d, J=8.7 Hz, 1H), 3.84 (d, J=9.0 Hz, 1H), 1.38 (s,9H).

Example 3 Synthesis of d₆-(S)-tert-butyl(1-((benzyloxy)amino)-3-hydroxy-3-methyl-1-oxobutan-2-yl)carbamate

N,N′-dicyclohexylcarbodiimide (5.9 g, 28.4 mmol, 1.1 equiv.) was addedto a solution ofd₆-(S)-2-((tert-butoxycarbonyl)amino)-3-hydroxy-3-methylbutanoic acid(6.0 g, 25.2 mmol, 1.0 equiv.) in N,N-dimethylformamide (80 ml) at r.t.followed by 1-hydroxybenzotriazole (3.8 g, 28.4 mmol, 1.1 equiv.). Theresulting mixture was stirred at r.t. for 30 min, and 98.0%O-benzylhydroxylamine hydrochloride (4.7 g, 28.6 mmol, 1.1 equiv.) wasadded followed by sodium bicarbonate (5.4 g, 64.5 mmol, 2.6 equiv.). Thereaction mixture was stirred at rt for 24 h and filtered through aCelite pad, washed with ethyl acetate (2×50 mL), and concentrated underreduced pressure at 40-50° C. The residue was diluted withdichloromethane (170 mL), loaded to a silica gel column (40 g, 30-40%ethyl acetate/hexanes) to give the title compound as a semi-solid afterelution. The semi-solid was sonicated with ethyl acetate andconcentrated on an oil pump for 2 h to give a white solid (7.5 g, 87%).¹HNMR (300 MHz, CDCl₃), δ 9.11 (s, 1H), 7.38-7.36 (m, 5H), 5.55 (d,J=8.7 Hz, 1H), 4.90 (s, 2H), 3.66 (d, J=8.7 Hz, 1H), 1.43 (s, 9H).

Example 4 Synthesis of(S)-tert-butyl(1-(benzyloxy)-2,2-dimethyl-d₆-4-oxoazetidin-3-yl)carbamate

Sulfur trioxide pyridine complex (1.8 g, 11.3 mmol, 1.4 equiv.) wasadded to a solution of d₆-(S)-tert-butyl(1-((benzyloxy)amino)-3-hydroxy-3-methyl-1-oxobutan-2-yl)carbamate (2.7g, 7.9 mmol, 1.0 equiv.) in pyridine (27 ml) at 0° C. in portions andthe mixture was stirred for 2 h. The pyridine was removed in vacuo andthe residue was triturated with diethyl ether/hexanes (1:10, 50 mL) toremove the major portion of pyridine. A solution of potassium carbonate(6.8 g, 49.0 mmol, 6.2 equiv.) in water (33 ml) and ethyl acetate (24ml) were added to the solid intermediate. The resulting mixture washeated under reflux (100° C.) for 3 h. The organic layer was separated,the aqueous layer was extracted with ethyl acetate (2×, total 600 mL),the combined organic layers were washed with brine, dried over anhydroussodium sulfate, filtered and concentrated. The material was dissolved indichloromethane and load to a silica gel column (25 g, 30-40% ethylacetate/hexanes) to afford the title compound as a white powder afterelution (1.28 g, 50%). ¹HNMR (300 MHz, DMSO-d6), δ 7.73 (d, J=8.7 Hz,1H), 7.41-7.38 (m, 5H), 4.92 (s, 2H), 4.25 (d, J=8.7 Hz, 1H), 1.38 (s,9H).

Example 5 Synthesis of (S)-tert-Butyl(1-hydroxy-2,2-dimethyl-d₆-4-oxoazetidin-3-yl)carbamate

10.0% palladium on carbon (0.4 g, 0.4 mmol, 0.1 equiv.) (wet, ˜50%water) was added to a solution of (S)-tert-butyl(1-(benzyloxy)-2,2-dimethyl-d₆-4-oxoazetidin-3-yl)carbamate (1.28 g, 3.9mmol, 1.0 equiv.) in methanol (25 mL) and the mixture was hydrogenatedunder hydrogen balloon at r.t. for 12 h. The reaction was then degassed,blanketed with argon and filtered through a Celite pad which was rinsedwith methanol (2×). The filtrate was concentrated in vacuo to give anoil which was triturated with 10% ether in hexanes, filtered and driedin vacuum to give the title compound as a white solid (quant.). ¹HNMR(300 MHz, DMSO-d6), δ 9.99 (brs, 1H), 7.70 (d, J=8.1 Hz, 1H), 4.19 (d,J=9.0 Hz, 1H), 1.39 (s, 9H).

Example 6 Synthesis of (S)-3-amino-2,2-dimethyl-d₆-4-oxoazetidin-1-ylhydrogen sulfate

Sulfur trioxide pyridine complex (0.75 g, 4.7 mmol, 1.2 equiv.) wasadded to a solution of (S)-tert-butyl(1-hydroxy-2,2-dimethyl-d₆-4-oxoazetidin-3-yl)carbamate (0.93 g, 3.9mmol, 1.0 equiv.) in pyridine (9 mL) at 0° C. The resulting mixture wasstirred at r.t. for 1.5 h and concentrated in vacuo to give thepyridinium salt as a foam that is used directly next step.

Pyridine(S)-3-((tert-butoxycarbonyl)amino)-2,2-dimethyl-d₆-4-oxoazetidin-1-ylsulfate (1.55 g, 3.9 mmol, 1.0 equiv.) was dissolved in 0.5 M potassiumphosphate monobasic (5.4 g, 39.8 mmol, 10.2 equiv.) in water (77 mL)solution. The mixture was extracted with dichloromethane (2×20 mL). Theaq. layer was cooled to 0° C. and 98.0% tetrabutylammonium hydrogensulfate (1.59 g, 4.6 mmol, 1.2 equiv.) was added to give a whitesuspension. The resulting mixture was stirred at r.t. for 1 h andextracted with dichloromethane (5×60 mL). The combined dichloromethanelayers were dried over anhydrous magnesium sulfate, filtered andconcentrated in vacuo to give the tetrabutylammonium salt which was useddirectly without purification.

Tetrabutylammonium(S)-3-((tert-butoxycarbonyl)amino)-2,2-dimethyl-d₆-4-oxoazetidin-1-ylsulfate (2.19 g, 3.9 mmol, 1.0 equiv.) was transferred to a pear shapedflask (250 mL), the residue dissolved in formic acid (6 ml) and theresulting solution was stirred at rt for 3 h. A white precipitate formedand the mixture was stirred at rt for an additional 2 h, dichloromethanewas added and the mixture was placed in a −20° C. refrigerator for 3days. The resulting solid was filtered and rinsed with colddichloromethane to afford the title compound as a white solid (0.195 g,23% over three steps). ¹HNMR (300 MHz, DMSO-d6), δ 8.76 (brs, 2H), 4.16(s, 1H).

Example 7 Synthesis of (S,Z)-tert-butyl2-(((2-((2,2-dimethyl-d₆-4-oxo-1-(sulfooxy)azetidin-3-yl)amino)-2-oxo-1-(2-(tritylamino)thiazol-4-yl)ethylidene)amino)oxy)acetate

N,N′-dicyclohexylcarbodiimide (0.21 g, 1.0 mmol, 1.1 equiv.) was addedto a solution of(Z)-2-((2-(tert-butoxy)-2-oxoethoxy)imino)-2-(2-(tritylamino)thiazol-4-yl)aceticacid (0.531 g, 1.0 mmol, 1.1 equiv.) in N,N-dimethylformamide (7 ml) atr.t. followed by 1-hydroxybenzotriazole (0.14 g, 1.0 mmol, 1.1 equiv.).The resulting mixture was stirred at rt for 30 min, and(S)-3-amino-2,2-dimethyl-d₆-4-oxoazetidin-1-yl hydrogen sulfate (0.192g, 0.9 mmol, 1.0 equiv.) was added followed by sodium bicarbonate (0.298g, 3.6 mmol, 4.0 equiv.). The resulting mixture was stirred at r.t.overnight and concentrated in vacuo at 40° C. (triturated with methanol,dichloromethane) to dryness. The residue was dissolved indichloromethane and loaded to a silica gel column (25 g, 5-10%MeOH/dichloromethane) to afford the title compound (0.65 g, 99%). ¹HNMR(300 Hz, CD₃OD), δ 7.36-7.26 (m, 15H), 6.82 (s, 1H), 4.74 (s, 1H, 4.61(s, 2H), 1.49 (s, 9H).

Example 8 Synthesis of TFA salt of(S,Z)-2-(((1-(2-aminothiazol-4-yl)-2-((2,2-dimethyl-d₆-4-oxo-1-(sulfooxy)azetidin-3-yl)amino)-2-oxoethylidene)amino)oxy)aceticacid

(S,Z)-tert-butyl2-(((2-(((2,2-dimethyl-d₆-4-oxo-1-(sulfooxy)azetidin-3-yl)amino)-2-oxo-1-(2-(tritylamino)thiazol-4-yl)ethylidene)amino)oxy)acetate(0.147 g, 0.2 mmol, 1.0 equiv.) and anisole (5 ul, 0.04 mmol, 0.2equiv.) and dry dichloromethane (0.7 ml) were charged into a flask witha stir bar under argon. The suspension was cooled to 0° C. andtrifluoroacetic acid (0.7 ml, 9.4 mmol, 47.6 equiv.) was added dropwisewithin 5 min. The suspension became a yellow solution once TFA wasadded. The ice-bath was allowed to warm to up to 15° C. within 4 h. Thereaction mixture was cooled to 0° C. with an ice-bath and cold deionizedwater (2 mL) was added dropwise. After separation, the aqueous layer waslyophilized in a 25 mL round bottom flask for 3 days. The resultinglight yellow solid was added to acetonitrile and DCM and sonicated togive a white suspension. The solid was filtered and then transferredback to the original flask and freeze-dried overnight to give a fluffywhite solid (84 mg, 76%).

Example 9 Removal of TFA

(S,Z)-2-(((1-(2-aminothiazol-4-yl)-2-(((2,2-dimethyl-d₆-4-oxo-1-(sulfooxy)azetidin-3-yl)amino)-2-oxoethylidene)amino)oxy)aceticacid (unspecified amount) was dissolved in D₂O (650 uL) and analyzed by¹H and ¹⁹F NMR with TFE (3 uL) and then transferred to a 20 mL vial andcooled to 0° C. and glacial acetic acid (1 mL) was added. The resultingsolution was stirred at 0° C. for 30 min The resulting mixture waslyophilized for 2 days. The process was repeated eight times to affordthe title compound (21.8 mg, 87.2%). TFA 4.6% by weight determined by ¹Hand ¹⁹F NMR. There were no visible acetic acid protons by ¹H NMR. ¹HNMR(300 MHz, D₂O), δ 7.07 (s, 1H), 4.89 (s, 1H), 4.73 (s, 2H, overlappedwith water), ¹⁹FNMR (282 MHz, D₂O), δ −75.56. LCMS: [M+1]⁺, 444.4.

Example 10 Synthesis of(R)-tert-butyl(3-hydroxy-1-(methoxy(methyl)amino)-1-oxopropan-2-yl)carbamate

Boc-D-Serine (25.5 g, 124.5 mmol, 1.0 equiv.) was dissolved intetrahydrofuran (112 mL) and N,O-dimethylhydroxylamine hydrochloride (14g, 143.3 mmol, 1.2 equiv.) in water (112 mL) was added. While cooling inan ice bath, 1N sodium hydroxide was added to bring the pH to 4.5, thensodium hydroxide (2.45 g, 61.2 mmol, 0.5 equiv.) in water (329 mL) wasadded to maintain pH 4.5 and a solution ofN-ethyl-N′-(3-dimethylaminopropyl)carbodiimide hydrochloride (27.8 g,145.0 mmol, 1.2 equiv.) in water (281 mL) was slowly added during 30 min(the reaction was monitored by pH meter). After stirring for 20 h atr.t., the solution was saturated with sodium chloride and extracted 3×with ethyl acetate (201 mL). Combined organic extracts were concentratedto give a crude solid. The solid was dissolved in ethyl acetate with thehelp of the heating and hexanes were added to give a light orangesolution. As the solution cooled, small colorless crystals startedforming. The solution was allowed to stand at r.t. for 30 min beforebeing placed in an ice-bath for 30 min The resulting colorless crystalswere filtered and rinsed with 10% ethyl acetate/hexanes and dried in airto afford the title compound as a shiny snow-flakes like solid (16.48g). The filtrate was concentrated and recrystallized to give the secondcrop (9.03 g) as a white solid. (total 25.5 g, 83%). ¹HNMR (300 MHz,CDCl₃), δ 5.56 (brs, 1H), 4.80 (brs, 1H), 3.83-3.78 (m, 5H), 3.23 (s,3H), 2.52 (brs, 1H), 1.45 (s, 9H).

Example 11 Synthesis of (R)-tert-butyl4-(methoxy(methyl)carbamoyl)-2,2-dimethyloxazolidine-3-carboxylate

A suspension of (R)-tert-butyl(3-hydroxy-1-(methoxy(methyl)amino)-1-oxopropan-2-yl)carbamate (49 g,197.4 mmol, 1.0 equiv.) in dry benzene (900 ml) was treated with2,2-dimethoxypropane (200 ml, 1626.5 mmol, 8.2 equiv.) and pyridiniump-toluenesulfonate (5 g, 20.0 mmol, 0.1 equiv.) and a Dean-Stark trapwas attached on a 2 L round bottom flask. The mixture was refluxed (75°C.) for 1 h without distillation and then the MeOH-benzene azeotrope wasslowly distilled at 80° C. during 0.5 h (11 mL, ¹HNMR indicated methanoland benzene and small amount of 2,2-dimethoxypropane), oil bath was thenheated to 85° C. and stirred for total 5 h with distillation (200 mL).TLC (50% ethyl acetate/hexanes, KMnO₄ stained, R_(f) of product, 0.52,starting material, 0.15) indicated the reaction was complete. Aftercooling, hexanes were added and the solution stored in a refrigeratorovernight. The resulting solid was filtered off and saturated aq. NaHCO₃was added. Extraction with ethyl acetate, followed by washing of theorganic extracts with brine, drying over anhydrous sodium sulfate,filtering and concentration gave crude product as an oil to which wasadded hexanes and the resulting orange solid filtered off. The filtratewas concentrated to give white solid, which was filtered and rinsed withhexanes and dried in air to give the product (43 g). The white solid wasdried in a vacuum oven for 2 h. The filtrate was concentrated andhexanes were added. The resulting solid was filtered and gave the secondcrop of the title product as a white solid. (total 56 g, 98%). LCMS:[M+1]⁺5 , 289.0. ¹HNMR (300 Hz, DMSO-d6), δ 4.72-4.68 (m, 1H), 4.21-4.14(m, 1H), 3.85-3.79 (m, 1H), 3.68-3.66 (m, 3H), 3.13-3.11 (m, 3H), 1.54(s, 3H), 1.45-1.41 (m, 6H), 1.33 (s, 6H).

Example 12 Synthesis of (R)-tert-butyl4-d₃-acetyl-2,2-dimethyloxazolidine-3-carboxylate

To a solution of (R)-tert-butyl4-(methoxy(methyl)carbamoyl)-2,2-dimethyloxazolidine-3-carboxylate (22g, 76.3 mmol, 1.0 equiv.) in dry tetrahydrofuran (63 mL) was addeddropwise d₃-Methylmaginesium iodide (91 ml, 91.6 mmol, 1.2 equiv.) whichhad been transferred to a 100 mL dropping funnel under argon atmospherevia canula at r.t. within 15 min. After stirring at r.t. for 1 h, asmall aliquot was quenched with saturated ammonium chloride solution at0° C. TLC (50% ethyl acetate/hexanes) and LCMS indicated the startingmaterial was all consumed. The reaction was cooled to 0° C. and quenchedwith saturated ammonium chloride solution and extracted with ethylacetate (3×). The combined organics were washed with brine, dried overanhydrous sodium sulfate, filtered and concentrated to give a yellowoil. The crude material was dissolved in dichloromethane and loaded to asilica gel (120 g, 0-70% ethyl acetate/hexanes) to afford the titlecompound as a pale yellow oil (16.24 g, 86%). ¹HNMR (300 MHz, DMSO-d6),δ 4.41 (dd, J=7.5, 3.0 Hz, 1H), 4.14-4.08 (m, 1H), 3.94 (dd, J=9.8, 2.5Hz, 1H), 1.54 (s, 3H), 1.42 (s, 7H), 1.33 (s, 5H). (Ref: Journal ofMedicinal Chemistry, 57(14), 5935-5948; 2014)

Example 13 Synthesis of (R,S)-tert-butyl4-(2-hydroxypropan-2-yl-2-methyl-2-d₃-methyloxazolidine-3-carboxylate

Methylmagnesiumchloride (50 ml, 150.4 mmol, 3.0 equiv.) in THF was addeddropwise to a cooled solution of (R)-tert-butyl4-d₃-acetyl-2,2-dimethyloxazolidine-3-carboxylate (12.4 g, 50.1 mmol,1.0 equiv.) in tetrahydrofuran (60 mL) in a 500 mL round bottom flask at−78° C. via dropping funnel within 15 min After 15 min, the temperaturewas allowed to rise slowly to 5° C. during 3 h. The mixture was cooledto 0° C. and quenched with saturated NH₄Cl, extracted with ethyl acetateand purified by chromatography. The crude light yellow oil was dissolvedin dichloromethane and loaded to a silica gel column (120 g, 10-20%ethyl acetate/hexanes) to give pure product as a pale yellow oil (12.14g, 91%). The methyl group from the two different isomers overlapped andthe ratio of isomers could not be determined by ¹H NMR. ¹HNMR (300 MHz,CDCl₃), δ 5.26 (brs, 1H), 4.01-3.98 (m, 2H), 3.79 (brs, 1H), 1.59 (s,3H), 1.50 (s, 12H), 1.18 (s, 3H).

Example 14 Synthesis oftert-butyl((2R,3S)-1,3-dihydroxy-3-d₃-methylbutan-2-yl)carbamate

A solution of (R,S)-tert-butyl4-(2-hydroxypropan-2-yl)-2-methyl-2-d₃-methyloxazolidine-3-carboxylate(12 g, 45.7 mmol, 1.0 equiv.) in methanol (405 mL) was treated withp-toluenesulfonic acid hydrate (0.87 g, 4.6 mmol, 0.1 equiv.). Thesolution was stirred for 70 min. at r.t. LCMS indicated presence ofstarting material and the reaction was allowed to continue to stir atr.t. for additional 30 min. TLC (20% ethyl acetate/hexanes) indicatedall starting material was consumed and TLC (50% ethyl acetate/hexanes)shoed the desired product at R_(f):0.6. The reaction was quenched withsaturated aqueous NaHCO₃ at r.t. and concentrated in vacuo in order toremove most of the methanol. The concentrated mixture was diluted withsaturated brine (and solid salt) and extracted with ethyl acetate (3×100mL). The organic extracts were combined and dried over anhydrous sodiumsulfate and concentrated to give pure product as a colorless oil. Theoil was dissolved in DICHLOREMETHANE and loaded on a silica gel column(120 g, 0-100% ethyl acetate/hexanes) to afford the title compound as aclear oil (quant.). LCMS indicated desired mass [M+1]⁺, 223.0. Ratio oftwo isomers:desired:undesired=2.61:1 by ¹H NMR. ¹HNMR (300 MHz,DMSO-d6), δ 6.21 (d, J=7.5 Hz, 1H), 4.42 (t, J=5.4 Hz, 1H), 4.31 (s,1H), 3.66-3.62 (m, 1H), 1.38 (s, 9H), 0.97 (s, 3H).

Example 15 Synthesis of(2S,3S)-2-((tert-butoxycarbonyl)amino)-3-hydroxy-3-d₃-methylbutanoicacid

TEMPO (0.71 g, 4.5 mmol, 0.1 equiv.) was added to a mixture oftert-butyl ((2R,3S)-1,3-dihydroxy-3-d₃-methylbutan-2-yl)carbamate (10.1g, 45.4 mmol, 1.0 equiv.) in acetonitrile (230 mL) and sodium phosphatebuffer (182 ml, 127.3 mmol, 2.8 equiv.) and the resulting mixture washeated to 35° C. The mixture was then treated by simultaneous additionof 80.0% sodium chlorite (13 g, 116.1 mmol, 2.6 equiv.) solution inwater (39 mL) and 4 drops of a very dilute sodium hypochlorite (3 mL ofcommercial solution in 100 mL of water). The mixture was stirred at 35°C. for 3 days, LCMS indicated the reaction was complete and the reactionwas typically very dark. The reaction mixture was treated with citricacid (9.2 g, 47.8 mmol, 1.1 equiv.) (pH 3), saturated with sodiumchloride and extracted with ethyl acetate (200 ml) (3×). The organicextracts were combined and concentrated. The resulting residue wasdissolved into sodium carbonate solution 2M (307 ml) and the solutionwas extracted with ethyl acetate (2×). The aqueous layer was cooled to0° C., the pH was adjusted to 3.0 using 4 M solution of H₃PO₄ (307 mL topH 3) and the solution was saturated with sodium chloride (stirred atr.t. for 30 min). The resulting mixture was extracted with ethyl acetate(200 mL) (3×), the organic phases were combined, dried, filtered andconcentrated under reduced pressure to give the title compound as asemi-solid which was sonicated with diethyl ether and filtered to give awhite solid (9.59 g, 89%). The ratio of the two isomers was not be ableto be determined by ¹H NMR since the two methyl signals overlapped.¹HNMR (300 MHz, DMSO-d6), δ 6.51 (d, J=8.7 Hz, 1H), 4.65 (brs, 1H), 3.85(d, J=9.6 Hz, 1H), 1.38 (s, 9H), 1.14 (s, 3H).

Example 16 Synthesis oftert-butyl((2S,3S)-1-((benzyloxy)amino)-3-hydroxy-3-d₃-methyl-1-oxobutan-2-yl)carbamate

N,N′-dicyclohexylcarbodiimide (4.7 g, 23.0 mmol, 1.1 equiv.) was addedto a solution of(2S,3S)-2-((tert-butoxycarbonyl)amino)-3-hydroxy-3-d₃-methylbutanoicacid (4.93 g, 20.9 mmol, 1.0 equiv.) in N,N-dimethylformamide (65 ml) atrt followed by 1-hydroxybenzotriazole (3.1 g, 23.0 mmol, 1.1 equiv.).The resulting mixture was stirred at rt for 30 min, andO-benzylhydroxylamine hydrochloride (3.66 g, 23.0 mmol, 1.1 equiv.) wasadded followed by sodium bicarbonate (7.0 g, 83.5 mmol, 4.0 equiv.). Thereaction mixture was stirred at rt for 15 h. LCMS indicated the desiredmass and the mass of(Z)—N-(((benzylamino)oxy)(cyclohexylamino)methylene)cyclohexanamine([M+1]⁺, 330). TLC (30% ethyl acetate/hexanes) indicated all startingcarboxylic acid was consumed and the mixture was filtered through aCelite pad into a saturated sodium bicarbonate solution and washed withethyl acetate (2×20 mL). The filtrate was diluted with ethyl acetate(500 mL) and washed with water (2×), brine, dried over anhydrous sodiumsulfate, filtered and concentrated. The resulting residue was dissolvedin dichloromethane and loaded to a silica gel column (80 g, 30-40% ethylacetate/hexanes) to give the title compound as a colorless solid (5.48g, 77%). The ratio of the two isomers was not be able to be determinedby ¹H NMR since the two methyl signals were overlapped. ¹HNMR (300 MHz,DMSO-d6), δ 11.04 (s, 1H), 7.39-7.33 (m, 5H), 6.42 (d, J=9.3 Hz, 1H),4.79 (s, 2H), 4.62 (s, 1H), 3.76 (d, J=9.6 Hz, 1H), 1.39 (s, 9H), 1.06(s, 3H).

Example 17 Synthesis oftert-butyl((2R,3S)-1-(benzyloxy)-2-d₃-methyl-2-methyl-4-oxoazetidin-3-yl)carbamate

98.0% sulfur trioxide pyridine complex (4.6 g, 28.5 mmol, 2.0 equiv.)was added to a solution of tert-butyl((2S,3S)-1-((benzyloxy)amino)-3-hydroxy-3-d₃-methyl-1-oxobutan-2-yl)carbamate(4.87 g, 14.3 mmol, 1.0 equiv.) in pyridine (48 mL) at 0° C. inportions, the ice-bath was removed and the mixture was stirred at r.t.for 4 h at which point the suspension became a clear solution. Crude ¹HNMR indicated the reaction was complete. Pyridine was removed in vacuoand the residue was triturated with diethyl ether/hexanes (1:10, 50 mL)to remove the major portion of pyridine at 30° C. with the help of anoil pump. A solution of potassium carbonate (12 g, 87.8 mmol, 6.2equiv.) in water (58 mL) and ethyl acetate (42 ml) were added to thesolid intermediate. The resulting mixture was heated under reflux (100°C.) for 16 h. The ethyl acetate layer was separated, the aqueous layerwas extracted with ethyl acetate (2×, total 160 mL) and the combinedorganic layers were washed with brine, dried over anhydrous sodiumsulfate, filtered and concentrated. The resultant material was dissolvedin dichloromethane and loaded on a silica gel column (80 g, 30-40% ethylacetate/hexanes) to afford the title compound as a white powder (2.59 g,56%). The ratio of two isomers:desired:undesired was 2.88:1 by ¹H NMR.¹HNMR (300 MHz, DMSO-d6), δ 7.72 (d, J=9.0 Hz, 1H), 7.43-7.38 (m, 5H),4.92 (s, 2H), 4.24 (d, J=8.7 Hz, 1H), 1.39 (s, 9H), 1.27 (s, 3H).

Example 18 Synthesis oftert-butyl((2R,3S)-2-d₃-methyl-1-hydroxy-2-methyl-4-oxoazetidin-3-yl)carbamate

10.0% palladium on carbon (1.1 g, 1.0 mmol, 0.1 equiv.) (wet, ˜50%water) was added to a solution of tert-butyl((2R,3S)-1-(benzyloxy)-2-d₃-methyl-2-methyl-4-oxoazetidin-3-yecarbamate(3.29 g, 10.2 mmol, 1.0 equiv.) in methanol (63 mL) and the mixture washydrogenated under balloon at r.t. for 12 h. TLC (30% ethylacetate/hexanes, Rf 0.2, stained with KMnO₄) indicated the reaction wascomplete. The reaction was degassed and blanket with argon and filteredthrough a Celite pad which was rinsed with methanol (2×). The filtratewas concentrated in vacuo to give the title compound as a white solid(quant.). The ratio of two isomers:desired:undesired=2.49:1 by ¹H NMR.¹HNMR (300 MHz, DMSO-d6), δ 7.68 (d, J=8.7 Hz, 1H), 4.19 (d, J=9.0 Hz,1H), 1.39 (s, 9H), 1.28 (s, 3H).

Example 19 Synthesis of(2R,3S)-3-amino-2-d₃-methyl-2-methyl-4-oxoazetidin-1-yl hydrogen sulfate

Sulfur trioxide pyridine complex (3.2 g, 20.3 mmol, 2.0 equiv.) wasadded to a solution of tert-butyl((2R,3S)-2-d₃-methyl-1-hydroxy-2-methyl-4-oxoazetidin-3-yl)carbamate(2.4 g, 10.2 mmol, 1.0 equiv.) in pyridine (24 mL) at 0° C. Theresulting mixture was stirred at rt for 1.5 h and concentrated in vacuoto give the title compound as a foam that is used directly next step.

Pyridinium(2R,3S)-3-((tert-butoxycarbonyl)amino)-2-d₃-methyl-2-methyl-4-oxoazetidin-1-ylhydrogen sulfate (4.0 g, 10.2 mmol, 1.0 equiv.) was dissolved in 0.5 MPotassium phosphate monobasic (13.9 g, 102.4 mmol, 10.1 equiv.) in water(199 mL) solution. The mixture was extracted with dichloromethane (2×10mL). The aqueous layer was cooled to 0° C. and 98.0% tetrabutylammoniumhydrogen sulfate (4.1 g, 11.8 mmol, 1.2 equiv.) was added to give awhite suspension. The resulting mixture was stirred at 0-5° C. for 1 hand extracted with dichloromethane (5×50 mL). The combineddichloromethane layers were dried over anhydrous magnesium sulfate,filtered and concentrated in vacuo to give the title compound which wasused directly without purification.

Tetrabutylammonium(2R,3S)-3-((tert-butoxycarbonyl)amino)-2-d₃-methyl-2-methyl-4-oxoazetidin-1-ylhydrogen sulfate (5.86 g, 10.6 mmol, 1.0 equiv.) was dissolved in formicacid (15 mL) and the resulting solution was stirred at r.t. for 2 daysto provide white precipitate. The mixture was added dichloromethane andcooled to 0° C. and the resulting solid was filtered and dried in airfor 10 min to afford the title compound as a white powder (1.35 g, 60%over three steps). The ratio of the two isomers was not be able to bedetermined since the two methyl signals were overlapped. ¹HNMR (300 MHz,DMSO-d6), δ 8.70 (brs, 2H), 4.15 (s, 1H), 1.42 (s, 3H).

Example 20 Synthesis of tert-butyl2-(((Z)-(2-(((2R,3S)-2-d₃-Methyl-2-methyl-4-oxo-1-(sulfooxy)azetidin-3-ylamino)-2-oxo-1-(2-(tritylamino)thiazol-4-yl)ethylidene)amino)oxy)acetate

N,N′-dicyclohexylcarbodiimide (0.426 g, 2.1 mmol, 1.1 equiv.) was addedto a solution of(Z)-2-((2-(tert-butoxy)-2-oxoethoxy)imino)-2-(2-(tritylamino)thiazol-4-yl)aceticacid (1.122 g, 2.1 mmol, 1.1 equiv.) in N,N-dimethylformamide (14 mL) atr.t. followed by 1-hydroxybenzotriazole (0.28 g, 2.1 mmol, 1.1 equiv.).The resulting mixture was stirred at r.t. for 30 min, and(2R,3S)-3-amino-2-d₃-methyl-2-methyl-4-oxoazetidin-1-yl hydrogen sulfate(0.4 g, 1.9 mmol, 1.0 equiv.) was added followed by sodium bicarbonate(0.63 g, 7.5 mmol, 4.0 equiv.). The resulting mixture was stirred at rtovernight and concentrated in vacuo at 30° C. to dryness. The residuewas dissolved in dichloromethane and loaded to a silica gel column (12g, 5-10% MeOH/dichloromethane) to afford the title compound (1.12 g,81%) which contains dichloromethane residue and small amount ofimpurities. The ratio of two isomers was not be able to be determinedsince one of the methyl signal was overlapped with tert-butyl signal by¹H NMR. ¹HNMR (300 MHz, DMSO-d6), δ 9.33 (d, J=7.5 Hz, 1H), 8.84 (s,1H), 7.36-7.20 (m, 15H), 6.71 (s, 1H), 4.54-4.50 (m, 3H), 1.42-1.40 (m,12H).

Example 21 Synthesis of2-(((Z)-(1-(2-aminothiazol-4-yl)-2-(((2R,3S)-2-d₃-2-methyl-4-oxo-1-(sulfooxy)azetidin-3-ylamino)-2-oxoethylidene)amino)oxy)aceticacid

Tert-butyl2-(((Z)-(2-(((2R,3S)-2-d₃-methyl-2-methyl-4-oxo-1-(sulfooxy)azetidin-3-yl)amino)-2-oxo-1-(2-(tritylamino)thiazol-4-yl)ethylidene)amino)oxy)acetate(1.1 g, 1.5 mmol, 1.0 equiv.) and anisole (32 ul, 0.3 mmol, 0.2 equiv.)and dry dichloromethane (6 mL) were charged in a flask with a stir barunder argon. The suspension was cooled to 0° C. and trifluoroacetic acid(6 ml, 80.8 mmol, 54.3 equiv.) was added dropwise within 7 min. Thesuspension became yellow once TFA was added. The ice-bath was allowed towarm to up to 10° C. within 4 h, LCMS indicated desired mass atretention time 3.89 min. The reaction was cooled to 0° C. with ice-bathand cold deionized water (12 mL) was added dropwise. After separation,the aqueous layer was lyophilized in a 250 mL round bottom flaskovernight. The resulting yellow material was added to dichloromethaneand acetonitrile and sonicated. The white solid was filtered andtransferred into a 20 mL vial with the help of water and acetonitrileand lyophilized overnight to afford a white fluffy solid (488 mg, 74%).LCMS: [M+1]⁺, 441.1.

Example 22 Removal of TFA

2-(((Z)-(1-(2-aminothiazol-4-yl)-2-(((2R,3S)-2-d₃-2-methyl-4-oxo-1-(sulfooxy)azetidin-3-yl)amino)-2-oxoethylidene)amino)oxy)aceticacid (unspecified amount) was dissolved in D₂O (700 uL) and acetic acid(4 ml) (1 mL×4) and the resulting clear solution was stirred at 0° C.for 30 min. The pale yellow clear solution was lyophilized overnight.The process was repeated six times to afford the final product (44 mg,88%). TFA 2.9% by weight after six cycles. Acetic acid content is ˜1% byweight (7% mol). The ratio between the desired and undesired isomers:2.66:1 by ¹H NMR. ¹HNMR (300 MHz, D₂O), δ 7.01 (s, 1H), 4.82 (s, 1H),4.67 (s, 2H, overlapped with water), 1.47 (s, 3H). ¹⁹FNMR (282 MHz,D₂O), δ −75.69. LCMS: [M+1]⁺, 441.3.

Example 23 Synthesis of (R)-tert-butyl4-acetyl-2,2-dimethyloxazolidine-3-carboxylate

A solution of (R)-tert-butyl4-(methoxy(methyl)carbamoyl)-2,2-dimethyloxazolidine-3-carboxylate (16.2g, 56.0 mmol, 1.0 equiv.) in dry tetrahydrofuran (46 mL) was cooled to−63° C., and slowly treated with a 1.6 M methyllithium solution indiethyl ether (70 mL). After stirring at −60° C. for 4 h, a smallaliquot was quenched with saturated ammonium chloride solution at −20°C., LCMS indicated desired mass along with starting material. TLC (50%ethyl acetate/hexanes, KMnO₄ stained showed three spots: R_(f), startingmaterial 0.125, product: 0.5, 0.75). The reaction mixture was cooled to−40° C. and quenched with saturated ammonium chloride slowly and allowedto warm to rt and extracted with diethyl ether (1×) and ethyl acetate(2×). Combined organic layers were washed with brine, dried overanhydrous sodium sulfate, filtered and concentrated to give a yellow oil(13.41 g). The crude material was dissolved in dichloromethane andloaded on a silica gel column (120 g, 0-100% ethyl acetate/hexanes) togive the desired product as a pale yellow oil (7.29 g, 53.3%). ¹HNMR(300 MHz, DMSO-d6), δ 4.41 (dd, J=7.5, 3.0 Hz, 1H), 4.15-4.08 (m, 1H),3.94 (dd, J=9.6, 2.7 Hz, 1H), 2.12 (s, 3H), 1.54 (s, 3H), 1.42-1.41 (m,6H), 1.33 (s, 6H).

Example 24 Synthesis of (R,R)-tert-butyl4-(2-hydroxypropan-2-yl)-2-methyl-2-d₃-methyloxazolidine-3-carboxylate

1 M solution of d₃-Methylmagnesium iodide (82 ml, 82.0 mmol, 2.8 equiv.)in diethyl ether was added dropwise to a cooled solution of(R)-tert-butyl 4-acetyl-2,2-dimethyloxazolidine-3-carboxylate (7.3 g,29.8 mmol, 1.0 equiv.) in tetrahydrofuran (37 mL) in a 250 mL roundbottom flask at −78° C., which resulted in formation of a whiteprecipitate. After 15 min, the temperature was allowed to rise slowly to0° C. during 3 h. The mixture was cooled to −60° C., quenched withsaturated NH₄Cl, extracted with Et₂O and purified by chromatography(ethyl acetate/hexanes: 10%-20%) to give as pure product a pale yellowoil (6.46 g, 83%). The ratio of two isomers was not able to bedetermined since methyl signal and tert-butyl signal overlapped. ¹HNMR(300 MHz, CDCl₃), δ 4.70-4.50 (brs, 1H), 4.04-3.99 (m, 1H), 3.87-3.70(m, 2H), 1.50 (s, 3H), 1.42 (s, 12H), 1.07-1.02 (d, J=15.3 Hz, 3H).

Example 25 Synthesis oftert-butyl((2R,3R)-1,3-dihydroxy-3-methylbutan-2-yl)carbamate

A solution of (R,R)-tert-butyl4-(2-hydroxypropan-2-yl)-2-methyl-2-d₃-methyloxazolidine-3-carboxylate(6.4 g, 24.4 mmol, 1.0 equiv.) in methanol (216 mL) was treated withp-toluenesulfonic acid hydrate (0.46 g, 2.4 mmol, 0.1 equiv.). Thesolution was stirred for 100 min. at rt. The reaction was quenched withsaturated aq. NaHCO₃ at rt, and concentrated in vacuo in order to removemost methanol. The concentrated mixture was diluted with saturated brine(and solid salt) and extracted with ethyl acetate (3×100 mL). Theorganic extracts were combined and dried over anhydrous sodium sulfate,concentrated to give as a colorless oil (4.89 g, 90%). LCMS: [M+1]⁺,223.0. The ratio between the desired and undesired isomers: 4.82:1 by ¹HNMR. ¹HNMR (300 MHz, CDCl₃), δ 5.63 (m, 1H), 4.05-4.01 (m, 1H),3.83-3.78 (m, 1H), 3.45 (m, 1H), 2.62-2.55 (m, 2H), 1.45 (s, 9H), 1.35(s, 3H).

Example 26 Synthesis of(2S,3R)-2-((tert-butoxycarbonyl)amino)-3-hydroxy-3-methylbutanoic acid

TEMPO (0.35 g, 2.2 mmol, 0.1 equiv.) was added to a mixture oftert-butyl ((2R,3R)-1,3-dihydroxy-3-methylbutan-2-yl)carbamate (4.89 g,22.0 mmol, 1.0 equiv.) in acetonitrile (111 mL) and sodium phosphatebuffer (88 mL) and the resulting mixture was heated to 35° C. Themixture was then treated by simultaneous addition of 80.0% sodiumchlorite (6.4 g, 56.2 mmol, 2.5 equiv.) solution in water (18 mL) and 4drops of a very dilute sodium hypochlorite (3 mL of commercial solutionin 100 mL of water). The mixture was stirred at 35° C. overnight, andLCMS indicated there was no reaction. TEMPO (0.35 g, 2.2 mmol, 0.1equiv.) and sodium hypochlorite (0.44 ml, 7.1 mmol, 0.3 equiv.) wasadded and the reaction was allowed to stir at 35° C. for additional 8 h.LCMS indicated the reaction was incomplete with mostly startingmaterial. The reaction was allowed to stir at 35° C. for additional 20 hand then r.t. for another day. LCMS indicated the reaction was completeand the reaction mixture was dark colored. The reaction mixture wastreated with citric acid (4.44 g, 23.1 mmol, 1.0 equiv.) (pH 3),saturated with sodium chloride and extracted with ethyl acetate (130 ml)(3×). The organic extracts were combined and concentrated. The resultingresidue was dissolved into sodium carbonate solution 2M (220 ml, 440.1mmol, 19.7 equiv.) and the solution was extracted with ethyl acetate(2×). The aqueous layer was cooled to 0° C., the pH was adjusted to 3.0using 4 M solution of H₃PO₄ (220 mL to pH 3) and the solution wassaturated with sodium chloride (stirred at rt for 30 min). The resultingmixture was extracted with ethyl acetate (130 ml) (3×), the organicphases were combined, dried, filtered and concentrated under reducedpressure to give the title compound as a semi-solid which was trituratedwith diethyl ether to give a white solid (5 g, 96%). The ratio of twoisomers was not able to be determined by ¹H NMR since two methyl signalsoverlapped. ¹HNMR (300 MHz, DMSO-d6), δ 6.51 (d, J=8.7 Hz, 1H), 3.85 (d,J=9.0 Hz, 1H), 1.39 (s, 9H), 1.16 (s, 3H).

Example 27 Synthesis oftert-butyl((2S,3R)-1-((benzyloxy)amino)-3-hydroxy-d₃-methyl-1-oxopentan-2-yl)carbamate

N,N′-dicyclohexylcarbodiimide (4.6 g, 22.1 mmol, 1.1 equiv.) was addedto a solution of(2S,3R)-2-((tert-butoxycarbonyl)amino)-3-hydroxy-3-methylbutanoic acid(4.68 g, 19.8 mmol, 1.0 equiv.) in N,N-dimethylformamide (62 mL) at r.t.followed by 1-hydroxybenzotriazole (2.98 g, 22.1 mmol, 1.1 equiv.). Theresulting mixture was stirred at rt for 30 min, andO-benzylhydroxylamine hydrochloride (3.6 g, 22.3 mmol, 1.1 equiv.) wasadded followed by sodium bicarbonate (6.7 g, 80.3 mmol, 4.0 equiv.). Thereaction mixture was stirred at rt for 15 h. LCMS indicated the desiredmass and the mass of(Z)—N-(((benzylamino)oxy)(cyclohexylamino)methylene)cyclohexanamine([M+1]⁺, 330. TLC (30% EA/Hex) indicated all starting carboxylic acidwas consumed and the mixture was filtered through a Celite pad into asaturated sodium bicarbonate solution, washed with ethyl acetate (2×20mL), the filtrate was diluted with ethyl acetate (200 mL) and washedwith water (2×), brine, dried over anhydrous sodium sulfate, filteredand concentrated and co-evaporated with toluene. The resulting residuewas dissolved in dichloromethane and loaded to a silica gel column (80g, 30-40% ethyl acetate/hexanes) to give the title compound as acolorless solid (3.48 g, 51%). The ratio of two isomers was not able tobe determined by ¹H NMR since two methyl signals overlapped. ¹HNMR (300MHz, DMSO-d6), δ 11.04 (s, 1H), 7.39-7.35 (m, 5H), 6.42 (d, J=8.7 Hz,1H), 4.79 (s, 2H), 4.62 (s, 1H), 3.76 (d, J=9.3 Hz, 1H), 1.39 (s, 9H),1.09 (s, 3H).

Example 28 Synthesis oftert-butyl((2S,3S)-1-(benzyloxy)-2-d₃-methyl-2-methyl-4-oxoazetidin-3-yl)carbamate

Sulfur trioxide pyridine complex (2.3 g, 14.4 mmol, 1.4 equiv.) wasadded to a solution of tert-butyl((2S,3R)-1-((benzyloxy)amino)-3-hydroxy-3-d₃-methyl-1-oxobutan-2-yl)carbamate(3.48 g, 10.2 mmol, 1.0 equiv.) in pyridine (35 mL) at 0° C. in portionsand the mixture was stirred for 2 h. Pyridine was removed in vacuo andthe residue was triturated with diethyl ether/hexanes (1:10, 50 mL) toremove the major portion of the pyridine. A solution of potassiumcarbonate (8.7 g, 62.7 mmol, 6.1 equiv.) in water (42 mL) and ethylacetate (30 ml) were added to the solid intermediate. The resultingmixture was heated under reflux (95° C. oil bath) for 6 h. The ethylacetate layer was separated and the aqueous layer was extracted withethyl acetate (2×, total 160 mL) and the combined organic layers werewashed with brine, dried over anhydrous sodium sulfate, filtered andconcentrated. The crude material was dissolved in dichloromethane andloaded on a silica gel column (40 g, 30-40% ethyl acetate/hexanes) toafford the title compound as a white powder (1.14 g, 35%). The ratiobetween the desired and undesired isomers: 4.25:1 by ¹H NMR. ¹HNMR (300MHz, DMSO-d6), δ 7.72 (d, J=9.0 Hz, 1H), 7.41-7.39 (m, 5H), 4.92 (s,2H), 4.24 (d, J=8.7 Hz, 1H), 1.39 (s, 9H), 1.27 (s, 0.61H), 1.05 (s,2.59H).

Example 29 Synthesis oftert-butyl((2S,3S)-2-d₃-methyl-1-hydroxy-2-methyl-4-oxoazetidin-3-yl)carbamate

10.0% palladium on carbon (0.38 g, 0.4 mmol, 0.1 equiv.) (wet, ˜50%water) was added to a solution of tert-butyl((2S,3S)-1-(benzyloxy)-2-d₃-methyl-2-methyl-4-oxoazetidin-3-yecarbamate(1.140 g, 3.5 mmol, 1.0 equiv.) in methanol (22 mL) and the mixture washydrogenated under balloon for 12 h. TLC (30% ethyl acetate/hexanes, Rf0.2, stained with KMnO₄) indicated the reaction was complete. Thereaction was degassed, blanketed with argon and filtered through aCelite pad which was rinsed with methanol (2×). The filtrate wasconcentrated in vacuo to give the title compound as a white solid (0.81g, 98%). The ratio between the desired and undesired isomers: 4.45:1 by¹H NMR. ¹HNMR (300 MHz, DMSO-d6), δ 9.98 (brs, 1H), 7.68 (d, J=8.1 Hz,1H), 4.19 (d, J=9.0 Hz, 1H), 1.39 (s, 9H), 1.10 (s, 3H).

Example 30 Synthesis of(2S,3S)-3-amino-2-d₃-methyl-2-methyl-4-oxoazetidin-1-yl hydrogen sulfate

Sulfur trioxide pyridine complex (0.67 g, 4.2 mmol, 1.2 equiv.) wasadded to a solution of tert-butyl((2S,3S)-2-d₃-methyl-1-hydroxy-2-methyl-4-oxoazetidin-3-yl)carbamate(0.81 g, 3.5 mmol, 1.0 equiv.) in pyridine (8 mL) at 0° C. The resultingmixture was stirred at rt for 1.5 h and concentrated in vacuo to givethe title compound as a foam that is used directly next step.

Pyridinium(2S,3S)-3-((tert-butoxycarbonyl)amino)-2-d₃-methyl-2-methyl-4-oxoazetidin-1-ylsulfate (1.36 g, 3.5 mmol, 1.0 equiv.) was dissolved in 0.5 M potassiumphosphate monobasic (4.8 g, 34.9 mmol, 10.0 equiv.) in water (68 ml)solution. The mixture was extracted with dichloromethane (2×10 mL). Theaqueous layer was cooled to 0° C. and 98.0% tetrabutylammonium hydrogensulfate (1.4 g, 4.0 mmol, 1.1 equiv.) was added to give a whitesuspension. The resulting mixture was stirred at 0-5° C. for 1 h andextracted with dichloromethane (5×50 mL). The combined dichloromethanelayers were dried over anhydrous magnesium sulfate, filtered andconcentrated in vacuo to give the title compound which was used directlywithout purification.

(2S,3S)-3-((tert-butoxycarbonyl)amino)-2-d₃-methyl-2-methyl-4-oxoazetidin-1-ylhydrogen sulfate (1.9 g, 6.1 mmol, 1.0 equiv.) was dissolved in formicacid (5 mL) and the resulting solution was stirred at r.t. for 5 h. Nowhite precipitate formed. The mixture was added to dichloromethane,which resulted in formation of a white precipitate, and was stored at−20° C. for two days. The resulting solid was filtered but the solidblocked the filter paper and hexanes were added and semi-solid wasdissolved in methanol and concentrated. The resulting solid wastriturated with dichloromethane and dried under vacuum for 1 h andrinsed with cold dichloromethane to afford the title compound as a whitesolid (107 mg, 33%). The ratio of two isomers was not able to bedetermined since two methyl signals overlapped by ¹H NMR. ¹HNMR (300MHz, DMSO-d6), δ 8.69 (brs, 2H), 4.17 (s, 1H), 1.41 (s, 3H).

Example 31 Synthesis of tert-butyl2-(((Z)-(2-(((2S,3S)-2-d₃-methyl-2-methyl-4-oxo-1-(sulfooxy)azetidin-3-ylamino)-2-oxo-1-(2-(tritylamino)thiazol-4-yl)ethylidene)amino)oxy)acetate

N,N′-dicyclohexylcarbodiimide (0.06 g, 0.3 mmol, 1.1 equiv.) was addedto a solution of(Z)-2-((2-(tert-butoxy)-2-oxoethoxy)imino)-2-(2-(tritylamino)thiazol-4-yl)aceticacid(0.157 g, 0.3 mmol, 1.1 equiv.) in N,N-dimethylformamide (1.9 ml) atr.t. followed by 1-hydroxybenzotriazole (0.04 g, 0.3 mmol, 1.1 equiv.).The resulting mixture was stirred at rt for 30 min, and(2S,3S)-3-amino-2-d₃-methyl-2-methyl-4-oxoazetidin-1-yl hydrogen sulfate(0.056 g, 0.3 mmol, 1.0 equiv.) was added followed by sodium bicarbonate(0.088 g, 1.1 mmol, 4.0 equiv.). The resulting mixture was stirred at rtovernight and concentrated in vacuo at 30° C. (triturated with methanol,dichloromethane) to dryness. The residue was dissolved indichloromethane and loaded to a silica gel column (12 g, 5-10%MeOH/dichloromethane) to afford the title compound as a white solid (176mg, 90.7%). The ratio of two isomers was not able to be determined by ¹HNMR since the two methyl signals overlapped. ¹HNMR (300 MHz, DMSO-d6), δ9.32 (d, J=7.5 Hz, 1H), 8.84 (s, 1H), 7.36-7.20 (m, 15H), 6.71 (s, 1H),4.54-4.50 (m, 3H), 1.42 (s, 9H), 1.20 (s, 3H).

Example 32 Synthesis of2-(((Z)-(1-(2-aminothiazol-4-yl-2-(((2S,3S)-2-(d₃-methyl-2-methyl-4-oxo-1-(sulfooxy)azetidin-3-ylamino)-2-oxoethylidene)amino)oxy)aceticacid TFA salt

tert-Butyl2-(((Z)-(2-(((2S,3S)-2-d₃-methyl-2-methyl-4-oxo-1-(sulfooxy)azetidin-3-yl)amino)-2-oxo-1-(2-(tritylamino)thiazol-4-yl)ethylidene)amino)oxy)acetate(0.176 g, 0.2 mmol, 1.0 equiv.) and anisole (6 ul, 0.1 mmol, 0.2 equiv.)and dry dichloromethane (1 mL) were charged into a flask with a stir barunder argon. The suspension was cooled to 0° C. and trifluoroacetic acid(1 ml, 13.5 mmol, 56.5 equiv.) was added dropwise within 5 min. Thesuspension became yellow once TFA was added. The ice-bath was allowed towarm to up to 15° C. within 4 h. LCMS indicated desired mass as m/z 441.The reaction was cooled to 0° C. with ice-bath and cold deionized water(2 mL) was added dropwise. After separation, aqueous layer waslyophilized in a 25 mL round bottom flask for 3 days. The resultingyellow material was added DCM and acetonitrile and sonicated. The whitesolid was filtered and all material was transferred into a 20 mL vialwith the help of water and acetonitrile and lyophilized again to afforda white fluffy solid (90 mg, 68%). LCMS: [M+1]⁺, 441.1.

Example 33 Removal of TFA

2-(((Z)-(1-(2-aminothiazol-4-yl)-2-(((2S,3S)-2-(d₃-methyl-2-methyl-4-oxo-1-(sulfooxy)azetidin-3-yl)amino)-2-oxoethylidene)amino)oxy)aceticacid (25 mg) was dissolved in D₂O (650 uL) and analyzed by ¹H NMR and¹⁹F NMR with TFE (3 uL) and then transferred to a 20 mL vial and cooledto 0° C. and glacial acetic acid (1 mL) was added. The resultingsolution was stirred at 0° C. for 30 min. The resulting mixture waslyophilized overnight, the process was repeated eight times to affordthe title compound (21.5 mg, 86%). TFA 3.7% by weight and acetic acidcontent was barely visible by ¹H NMR. The ratio between the desired andundesired isomers: 4.98:1 by ¹H NMR. ¹HNMR (300 MHz, D₂O), δ 7.07 (s,1H), 4.89 (s, 1H), 4.70 (s, 2H, overlapped with water), 1.35 (s, 3H).¹⁹FNMR (282 MHz, D₂O), δ −75.55.

Example 34 Synthesis of(Z)-2-((2-(tert-butoxy)-2-oxoethoxy)imino)-2-(2-(tritylamino)thiazol-4-yl)aceticacid

Triethylamine (4.03 ml, 28.9 mmol, 1.2 equiv.) and triphenylmethylchloride (7.92 g, 28.4 mmol, 1.2 equiv.) were added to a suspension of98.0% ethyl 2-(2-aminothiazol-4-yl)glyoxylate (5.0 g, 24.5 mmol, 1.0equiv.) in dichloromethane (100 mL) at 0° C. The reaction mixture wasstirred for 15 min. and then allowed to warm to ambient temperature over3 h before the solvent was evaporated in vacuo. Water (100 mL) was addedand was extracted with ethyl acetate. The organic layer was washed withbrine, dried over anhydrous sodium sulfate and concentrated underreduced pressure. The crude material was purified by flash silicachromatography (80 g, 0-40% ethyl acetate/hexanes) to give a yellowsolid (10 g, 92.3%). LCMS: [M+1]⁺, 442.7. ¹HNMR (300 MHz, DMSO-d6), δ9.05 (s, 1H), 7.85 (s, 1H), 7.37-7.16 (m, 15H), 4.07-3.99 (m, 2H),1.18-1.10 (m, 3H).

Sodium hydroxide (37 mL of a 1.0 N solution in water) was added to asolution of ethyl 2-(2-(tritylamino)thiazol-5-yl)acrylate (10.000 g,22.6 mmol, 1.0 equiv.) in ethanol (90 mL) at 0° C. Tetrahydrofuran (25mL) was added and the reaction was allowed to warm to ambienttemperature over 3 h before the solvent was evaporated in vacuo. Theresulting residue was acidified with aq. hydrochloric acid (6.0 N) to pH2 and the product collected by suction filtration to yield a yellowsolid (9.03 g, 96.4%). ¹HINMR (300 MHz, DMSO-d6), δ 8.99 (s, 1H), 7.74(s, 1H), 7.33-7.20 (m, 15H).

A solution of 98.0% tert-butyl 2-(aminooxy)acetate (3.27 g, 21.8 mmol,1.0 equiv.) in methanol (100 mL) was treated with2-oxo-2-(2-(tritylamino)thiazol-5-yl)acetic acid (9.03 g, 21.8 mmol, 1.0equiv.) and stirred at ambient temperature for 3 h. The reaction mixturewas evaporated in vacuo. The crude material was purified by flash silicachromatography (0-15% MeOH/dichloromethane, 120 g) to give a beige solid(9.33 g). HPLC analysis, indicated that there were two peaks (one majorand the other one is minor) next to each other and not separable byflash column chromatography. Purified by prep-TLC (5% MeOH/DCM), theminor peak was isolated and concentrated. LCMS indicated the minor peakcompound was the isomer of the major peak. The beige solid wasrecrystallized in methanol to give the desired compound as a whitepowder (8.22 g, 69.4%). LCMS: [M+1]⁺, 554.0. ¹HNMR (300 MHz, DMSO-d6), δ8.82 (s, 1H), 7.33-7.17 (m, 15H), 6.82 (s, 1H), 4.51 (s, 2H), 1.39 (s,9H).

Example 35 IV Dosing of Tigemonam and(S,Z)-2-(((1-(2-aminothiazol-4-yl)-2-((2,2-dimethyl-d₆-4-oxo-1-(sulfooxy)azetidin-3-yl)amino)-2-oxoethylidene)amino)oxy)aceticacid

A cohort of three rats was IV dosed using 2 mg/kg cassette IV dosing.The cassette included 1 mg/kg of tigemonam and 1 mg/kg of gem-dimethyld₆ analog((S,Z)-2-(((1-(2-aminothiazol-4-yl)-2-((2,2-dimethyl-d₆-4-oxo-1-(sulfooxy)azetidin-3-yl)amino)-2-oxoethylidene)amino)oxy)aceticacid). Samples were withdrawn at various time points indicated below andprocessed using the following procedure. A 50 μL rat plasma sample wastransferred to a well of a 96 well-plate, 20 μL of 0.1 ng/μL of Losartan(internal standard) in acetonitrile was added and 300 μL ofacetonitrile/formic acid (100/2). The sample was then vortexed andcentrifuged at 300 rpm for 5 minutes. Then 200 μL of the supernatant wasmixed with 200 μL of double distilled water and was injected (5 μL) on aphenomex Polar-RP 80A column (75×2.0 mm), which was run at 30° C. at aflow rate of 600 μL/minute with a mobile phase A of acetonitrile/formicacid 100/0.02 and a mobile phase B of water/formic acid 100/0.02. Theeluate was analyzed by LC/MS/MS (electrospray) to provide the followingvalues of plasma concentration vs. time listed in Table 1, which werethen plotted to yield the graph illustrated in FIG. 1.

TABLE 1 Time a b (hours) (ng/mL) (ng/mL) r = b − a Ratio of b/a 0.1675262 5497 235 1.04 0.5 2030 2049 19 1.01 1 468 437 −31 .9 2 146 167 211.14 4 32 34 2 1.06 6 5 3 −2 .6 8 0 0 0 24 0 0 0a and b in Table 1 above refer to tigemonam and(S,Z)-2-((((1-(2-aminothiazol-4-yl)-2-((2,2-dimethyl-d₆-4-oxo-1-(sulfooxy)azetidin-3-yl)amino)-2-oxoethylidene)amino)oxy)aceticacid, respectively. Each time point above represents a value obtained byaveraging the measured plasma concentration of three rats. As can beseen from Table 1, the plasma levels of tigemonam (solid line in FIG. 1)and(S,Z)-2-(((1-(2-aminothiazol-4-yl)-2-((2,2-dimethyl-d₆-4-oxo-1-(sulfooxy)azetidin-3-yl)amino)-2-oxoethylidene)amino)oxy)aceticacid (dashed line in FIG. 1) were almost identical. Deuteration oftigemonam thus appears to have no effect on IV bioavailability.

Example 36 Oral Dosing of Tigemonam and(S,Z)-2-(((1-(2-aminothiazol-4-yl)-2-((2,2-dimethyl-d₆-4-oxo-1-(sulfooxy)azetidin-3-yl)amino)-2-oxoethylidene)amino)oxy)aceticacid

A cohort of three rats was orally dosed using 10 mg/kg cassette IVdosing. The cassette included 5 mg/kg of tigemonam and 5 mg/kg ofgem-dimethyl d₆ analog((S,Z)-2-(((1-(2-aminothiazol-4-yl)-2-((2,2-dimethyl-d₆-4-oxo-1-(sulfooxy)azetidin-3-yl)amino)-2-oxoethylidene)amino)oxy)aceticacid). Samples were withdrawn at various time points indicated below andprocessed using the following procedure. A 50 μL rat plasma sample wastransferred to a well of a 96 well-plate, 20 μL of 0.1 ng/μL of Losartan(internal standard) in acetonitrile was added and 300 μL ofacetonitrile/formic acid (100/2). The sample was then vortexed andcentrifuged at 300 rpm for 5 minutes. Then 200 μL of the supernatant wasmixed with 200 μL of double distilled water and was injected (5 μL) on aphenomex Polar-RP 80A column (75×2.0 mm), which was run at 30° C. at aflow rate of 600 μL/minute with a mobile phase A of acetonitrile/formicacid 100/0.02 and a mobile phase B of water/formic acid 100/0.02. Theeluate was analyzed by LC/MS/MS (electrospray) to provide the followingvalues of plasma concentration vs. time listed in Table 1, which werethen plotted to yield the graph illustrated in FIG. 2.

TABLE 2 Time a b (hours) (ng/mL) (ng/mL) r = b − a Ratio of b/a 0.1671434 1700 266 1.19 0.5 2923 3617 694 1.24 1 2121 2652 531 1.25 2 679 854175 1.26 4 114 169 55 1.48 C6 27 31 4 1.15 8 04 5 1 1.25 24 0 0 0a and b in Table 1 above refer to tigemonam and(S,Z)-2-(((1-(2-aminothiazol-4-yl)-2-((2,2-dimethyl-d₆-4-oxo-1-(sulfooxy)azetidin-3-yl)amino)-2-oxoethylidene)amino)oxy)aceticacid, respectively. Each time point above represents a value obtained byaveraging the measured plasma concentration of three rats. As can beseen from Table 1 and FIG. 2, the plasma level of(S,Z)-2-(((1-(2-aminothiazol-4-yl)-2-((2,2-dimethyl-d₆-4-oxo-1-(sulfooxy)azetidin-3-yl)amino)-2-oxoethylidene)amino)oxy)aceticacid (dashed line in FIG. 2) was at least about 20% greater than theplasma level of tigemonam (solid line in FIG. 2). The above resultsindicates thatS,Z)-2-(((1-(2-aminothiazol-4-yl)-2-((2,2-dimethyl-d₆-4-oxo-1-(sulfooxy)azetidin-3-yl)amino)-2-oxoethylidene)amino)oxy)aceticacid is orally absorbed at an increased level when compared withtigemonam. Deuteration of tigemonam, thus appears to increase oralbioavailability of the antibiotic. Other compounds may also be tested inthe above assay.

Example 37 Efficacy of(S,Z)-2-(((1-(2-aminothiazol-4-yl)-2-((2,2-dimethyl-d₆-4-oxo-1-(sulfooxy)azetidin-3-yl)amino)-2-oxoethylidene)amino)oxy)aceticacid Alone or in Combination with a Carbapenem Antibiotic in a MouseThigh Model of K. Pneumoniae or E. Coli, Infection

The following is an example of an animal assay which is used todemonstrate efficacy of compounds disclosed herein in treating infectionwith bacterial pathogens. Those of skill in the art will appreciate thatvariations in certain parameters may be made.

All animal experiments are performed under IACUC review with ethicalcommittee clearance. Mice used in this study are specific pathogen free.Mice are allowed to acclimatize for 7 days and weighted 20-25 g at thestart of the experiment. Mice are housed in sterile individualventilated cages. The mice are exposed at all times to HEPA filteredsterile air. Mice have free access to food and water (sterile) and havesterile aspen chip bedding (is changed every 3-4 days or as isappropriate). A. Klebsiella pneumoniae isolate or Escherichia coliisolate resistant to antibiotics is used throughout the study.

In this study, 6 mice are used in each treatment group. Mice arerendered temporarily neutropenic by immunosuppression withcyclophosphamide at 150 mg/kg four days before infection and 100 mg/kgone day before infection by intraperitoneal injection. Theimmunosuppression regime leads to neutropenia starting 24 hours postadministration which continues throughout the study. 24 hours post thesecond round of immunosuppression mice are infected with bacteriaintramuscularly into both lateral thigh muscles using approximately2.5×10 cfu/mouse thigh. Antibacterial treatment is initiated 2 hourspost infection and is administered by gavage 2-3 times per 24 h. Analiquot of 4% CHD-FA stock solution as adjusted to pH 5 with 10 M sodiumhydroxide solution is then diluted either 1:2 or 1:8 with 0.9% saline togive a dosing solution of 2% (200 mg/kg) and 0.5% (50 mg/kg) CHD-FArespectively. Twenty-four hours post infection, the clinical conditionof all animals is assessed prior to them being humanely euthanized.Animal weight is determined before both thighs are removed and weighedindividually. Individual thigh tissue samples are homogenized in icecold sterile phosphate buffered saline. Thigh homogenates are thenquantitatively cultured onto CLED agar and incubated at 37° C. for 24hrs and colonies are counted daily. Results of CFU/gr and mortality areused to establish the potency of(S,Z)-2-(((1-(2-aminothiazol-4-yl)-2-((2,2-dimethyl-d₆-4-oxo-1-(sulfooxy)azetidin-3-yl)amino)-2-oxoethylidene)amino)oxy)aceticacid vs comparator.

It is expected that animals treated with(S,Z)-2-(((1-(2-aminothiazol-4-yl)-2-((2,2-dimethyl-d₆-4-oxo-1-(sulfooxy)azetidin-3-yl)amino)-2-oxoethylidene)amino)oxy)aceticacid will have reduced CFU/gr and reduced mortality compared tountreated animals and animals treated with an equivalent amount oftigemonam.

All references, patents or applications, U.S. or foreign, cited in theapplication are hereby incorporated by reference as if written herein intheir entireties. Where any inconsistencies arise, material literallydisclosed herein controls. Such references are not admitted to be priorart relevant to analysis of novelty, obviousness, or inventive step.

From the foregoing description, one skilled in the art can easilyascertain the essential characteristics of this invention, and withoutdeparting from the spirit and scope thereof, can make various changesand modifications of the invention to adapt it to various usages andconditions.

1.-20. (canceled)
 21. A compound of structural Formula (III):

or pharmaceutically acceptable salts, hydrates and solvates thereof,wherein: X is —CD₃ or —CH₃; and Y is —CD₃ or —CH₃; provided that X and Yare not both —CH₃.
 22. The compound of claim 21, having the structure:


23. The compound of claim 21, having the structure:


24. The compound of claim 21, having the structure:


25. A pharmaceutical composition comprising a compound of claim 21 and apharmaceutically acceptable excipient.